Antimicrobial cationic peptides

ABSTRACT

A novel class of cationic peptides having antimicrobial activity is provided. Examples of such peptides include NH2-KWKSFIKKLTTAVKKVLTTGLPALIS-COOH(SEQ ID NO:1)and NH2-KWKSFIKKLTSAAKKVVTTAKPLISS-COOH.(SEQ ID NO:2)Also provided are methods for inhibiting the growth of bacteria utilizing the peptides of the invention. The peptides are particularly useful for inhibiting endotoxemia in a subject.

This application is a Continuation-in-Part application of U.S. patentapplication Ser. No. 08/460,464 filed on Jun. 2, 1995 now U.S. Pat. No.5,877,274.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to antimicrobial peptides andspecifically to a new class of antimicrobial cationic peptides referredto as bactolysins.

2. Description of Related Art

In 1981, the self-promoted uptake hypothesis was first proposed toexplain the mechanism of action of polycationic antibiotics inPseudomonas aeruginosa. According to this hypothesis, polycationsinteract with sites on the outer membranes of Gram-negative bacteria atwhich divalent cations cross-bridge adjacent lipopolysaccharidemolecules. Due to their higher affinity for these sites, polycationsdisplace the divalent cations and, since the polycations are bulkierthan the divalent cations, cause structural perturbations in the outermembrane. These perturbations result in increased outer membranepermeability to compounds such as the β-lactam antibiotic nitrocefin,the eukaryotic non-specific defense protein lysozyme and to hydrophobicsubstances. By analogy, molecules accessing this pathway are proposed topromote their own uptake.

It has been clearly demonstrated that the outer membranes ofGram-negative bacteria are semipermeable molecular "sieves" whichrestrict access of antibiotics and host defense molecules to theirtargets within the bacterial cell. Thus, cations and polycations whichaccess the self-promoted uptake system are, by virtue of their abilityto interact with and break down the outer membrane permeability barrier,capable of increasing the susceptibility of Gram-negative pathogenicbacteria to antibiotics and host defense molecules. Hancock and Wongdemonstrated that a broad range of such compounds could overcome thepermeability barrier and coined the name "permeabilizers" to describethem (Hancock and Wong, Antimicrob. Agents Chemother., 26:48, 1984).While self-promoted uptake and permeabilizers were first described forP. aeruginosa, they have now been described for a variety ofGram-negative bacteria.

Over the past decade, non-specific defense molecules have been describedin many animals, including insects and humans. One subset of thesemolecules have in common the following features: (a) they are smallpeptides, usually 15-35 amino acids in length, (b) they contain 4 ormore positively charged amino acid residues, either lysines orarginines, and (c) they are found in high abundance in the organismsfrom which they derive. Several of these molecules have been isolated,amino acid sequenced and described in the patent literature (e.g.,cecropins: WO8900199, WO 8805826, WO8604356, WO 8805826; defensins: EP193351, EP 85250, EP 162161, U.S. Pat. No. 4,659,692, WO 8911291).However, only limited amounts of these peptides can be isolated from thehost species. For example, Sawyer, et al., (Infect. Immm. 56:693, 1988)isolated 100-200 mg of rabbit neutrophil defensins 1 and 2 from 10⁹primed peritoneal neutrophils or lipopolysaccharide-elicited alveolarmacrophages (i.e., the numbers present in a whole animal).

The gene for human defensin has been cloned and sequenced, but nosuccessful expression has been demonstrated, as yet. Furthermore,production of these peptides using peptide synthesis technology producespeptides in limited amounts and is expensive when scaled up or when manyvariant peptides must be produced. Also, structural analysis isdifficult without specific incorporation of ¹⁵ N and ¹³ C tagged aminoacids which is prohibitively expensive using amino acid synthesistechnology.

There is a need to develop polypeptides having a broad range of potentantimicrobial activity against a plurality of microorganisms, includinggram negative bacteria, gram positive bacteria, fungi, protozoa, virusesand the like.

SUMMARY OF THE INVENTION

The present invention provides a novel class of cationic peptides,referred to as bactolysins, which have antimicrobial activity. Tworepresentative peptides are provided and include

                          (SEQ ID NO:1)                                           MBI 29, NH.sub.2 -KWKSFIKKLTTAVKKVLTTGLPALIS-COOH                             and                                                                                                 (SEQ ID NO:2)                                           MBI 26, NH.sub.2 -KWKSFIKKLTSAAKKVVTTAKPLISS-COOH,                        

analogs, derivatives and conservative variations thereof.

The invention also provides a method of inhibiting the growth ofbacteria comprising contacting the bacteria with an inhibiting effectiveamount of a peptide having an amino acid sequence of MBI 29 (SEQ IDNO:1) or MBI 26 (SEQ ID NO:2) alone, or in combination with anantibiotic. Classes of antibiotics which can be used for synergistictherapy with the peptides of the invention include aminoglycoside,penicillin, cephalosporine, fluoroquinolone, carbepenem, tetracyclineand macrolide.

In another embodiment, the invention provides a method of inhibiting anendotoxemia or sepsis associated disorder in a subject having or at riskof having such a disorder, comprising administering to the subject atherapeutically effective amount of a peptide of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a three-dimensional schematic representation of theconformational structure of CEME, MBI 29, and MBI 26.

FIG. 2 shows the axial projection of the α-helical conformation of eachpeptide in 2D.

FIG. 3 shows an inner membrane permeabilization assay for MBI 26.

FIG. 4 shows an inner membrane permeabilization assay for MBI 29.

FIG. 5a shows tumor necrosis factor (TNF) levels measured 6 hours afterthe addition of E. coli 0111:B4 LPS and CEME (ME), CEMA (MA), and MBI 29to macrophage cells. The data is from two separate assays.

FIGS. 5b and 5c show tumor necrosis factor (TNF) levels measured 6 hoursafter the addition of E. coli Bort LPS and E. coli 0111:B4 LPS and MBI29 to macrophage cells. The data is from two separate assays.

FIG. 6a shows tumor necrosis factor (TNF) levels measured 6 hours afterthe addition of E. coli Bort LPS and CEME (ME), CEMA (MA) and MBI 29 tomacrophage cells. The data is from two separate assays.

FIG. 6b shows tumor necrosis factor (TNF) levels measured 6 hours afterthe addition of P. aeruginosa LPS and MBI 26 to RAW macrophage cells.

FIG. 6c shows tumor necrosis factor (TNF) levels measured 6 hours afterthe addition of E. coli Bort LPS, E. coli 0111:B4 LPS and MBI 26 to RAWmacrophage cells.

FIGS. 7a and 7b show RAW cell TNF production after addition of E. coli0111:B4 LPS and the effect of the addition of MBI 29 and polymyxin B onTNF.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a novel class of cationic peptides,called bactolysins, which have antimicrobial activity and have theability to significantly reduce the level of lipopolysaccharide(LPS)-induced tumor necrosis factor (TNF). These peptides are useful forinhibiting microbial infection or growth, as well reducing the effectsof endotoxemia and are often synergistic with conventional antibioticsand/or lysozyme. In addition, such peptides are useful as antifungalagents, antitumor agents, or antiviral agents.

The term "antimicrobial" as used herein means that the peptides of thepresent invention inhibit, prevent, or destroy the growth orproliferation of microbes such as bacteria, fungi, viruses or the like.The term "antiviral" as used herein means that the peptides of thepresent invention inhibit, prevent or destroy the growth orproliferation of viruses or of virally-infected cells. The term"anti-tumor" as used herein means that the peptides of the presentinvention may be used to inhibit the growth of or destroy tumors. Theterm "antifungal" as used herein means that the peptides of the presentinvention may be used to inhibit the growth of or destroy fungi.

In a first embodiment, the invention provides an isolated antimicrobialpeptide having an amino acid sequence:

                         (SEQ ID NO:26)                                           NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2          R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 VLTTGLPALIS-COOH,                                          (SEQ ID NO:27)                                           NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2          R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 VVTTAKPLISS-COOH,                                          (SEQ ID NO:28)                                           NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2          R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 ILTTGLPALIS-COOH,                                          (SEQ ID NO:29)                                           NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2          R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 GGLLSNIVTSL-COOH,                     or                                                                                                 (SEQ ID NO:30)                                           NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2          R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 GPILANLVSIV-COOH                  

wherein R₁ is a hydrophobic amino acid residue and R₂ is a hydrophilicamino acid residue.

Examples of such peptides of the invention include but are not limitedto:

                          (SEQ ID NO:1)                                           NH.sub.2 -KWKSFIKKLTTAVKKVLTTGLPALIS-COOH(MBI 29),                                                  (SEQ ID NO:2)                                           NH.sub.2 -KWKSFIKKLTSAAKKVVTTAKPLISS-COOH(MBI 26),                                                  (SEQ ID NO:3)                                           NH.sub.2 -KWKSFIKNLTKGGSKILTTGLPALIS-COOH(MBI 201),                                                 (SEQ ID NO:4)                                           NH.sub.2 -KWKKFIKNLTKGGSKILTTGLPALIS-COOH(MBI 202),                                                 (SEQ ID NO:5)                                           NH.sub.2 -KWKSFIKNLEKVLKPGGLLSNIVTSL-COOH(MBI 490),                                                 (SEQ ID NO:6)                                           NH.sub.2 -KWKSFIKNLEKVLKKGPILANLVSIV-COOH(MBI 491),                                                 (SEQ ID NO:7)                                           NH.sub.2 -KWKEFIKKLTTAVKKVLTTGLPALIS-COOH(MBI 492),                                                 (SEQ ID NO:8)                                           NH.sub.2 -KWKKFIKELQKVLAPGGLLSNIVTSL-COOH(MBI 493),                                                 (SEQ ID NO:9)                                           NH.sub.2 -KWKSFIKKLTSVLKKVVTTALPALIS-COOH(MBI 29A1),                                                (SEQ ID NO:10)                                          NH.sub.2 -KWKSFIKNLTKVLKKVVTTALPALIS-COOH(MBI 29A2),                                                (SEQ ID NO:11)                                          NH.sub.2 -KWKLFKKKGTGAVLTVLTTGLPALIS-COOH(MBI 29A3),                                                (SEQ ID NO:12)                                          NH.sub.2 -KWKSFIKKLTSVLKKVVTTAKPLISS-COOH,                                                          (SEQ ID NO:13)                                          NH.sub.2 -KKKSFIKLLTSAKVSVLTTAKPLISS-COOH,                                    and                                                                                                 (SEQ ID NO:14)                                          NH.sub.2 -KWKKFIKELQKVLKPGGLLSNIVTSL-COOH,                                

analogs, derivatives and conservative variations thereof, wherein thepeptides have antimicrobial activity. The peptides of the inventioninclude SEQ ID NO: 1-14, as well as the broader groups of peptideshaving hydrophilic and hydrophobic substitutions, and conservativevariations thereof.

The sequence KWKSFIKK (SEQ. ID NO:34), as found in the first 8 NH₂terminal amino acids of peptides in SEQ ID NO:1 and 2, is important forconferring antimicrobial activity. The presence of a positively chargedamino acid (lysine or arginine) at positions 11, 14, 15 and 21 are alsovery important in antimicrobial activity. A further enhancement ofantimicrobial activity can be achieved by making conservative changes inthe residues designated R₁ and R₂.

In another embodiment, the invention provides an isolated antimicrobialpeptide having an amino acid sequence:

    NH.sub.2 -KKWWRRR.sub.1 R.sub.1 R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 GPALSNV-COOH                                              (SEQ. ID NO:31)

wherein R₁ is a hydrophobic amino acid residue and R₂ is a hydrophilicamino acid residue. Examples of such peptides of the invention includebut are not limited to:

    NH.sub.2 -KKWWRRVLSGLKTGPALSVN-COOH,                                                                  (SEQ ID NO:15)                                        NH.sub.2 KKWWRRVLKGLSSGPALSNV-COOH,                                                                   (SEQ ID NO:16)                                        NH.sub.2 -KKWWRRALQALKNGPALSNV-COOH,                                                                  (SEQ ID NO:17)                                    

analogs, derivatives and conservative variations thereof, wherein thepeptides have antimicrobial activity. The peptides of the inventioninclude SEQ ID NO:15-17, as well as the broader groups of peptideshaving hydrophilic and hydrophobic substitutions, and conservativevariations thereof

In another embodiment, the invention provides an isolated antimicrobialpeptide having an amino terminal amino acid sequence:

    NH.sub.2 -KKWWRRX.                                         (SEQ. ID NO:32)

Preferably, the peptide is from about 20 to about 30 amino acids inlength and therefore X is from about 14 to about 24 amino acids. Mostpreferably the peptide has a sequence of

    NH.sub.2 -KKWWRRR.sub.1 R.sub.1 R.sub.2 GLKTAGPAIQSVLNK-COOH(SEQ. ID NO:35)

wherein R₁ is a hydrophobic amino acid residue and R₂ is a hydrophilicamino acid residue. Examples of such peptides of the invention includebut are not limited to:

                          (SEQ ID NO:18)                                          NH.sub.2 -KKWWRRVLSGLKTAGPAIQSVLNK-COOH (MBI 21A1),                           and                                                                                                 (SEQ ID NO:19)                                          NH.sub.2 -KKWWRRALQGLKTAGPAIQSVLNK-COOH (MBI 21A2),                       

analogs, derivatives and conservative variations thereof, wherein thepeptides have antimicrobial activity. The peptides of the inventioninclude SEQ ID NO: 18-19, as well as the broader groups of peptideshaving hydrophilic and hydrophobic substitutions, and conservativevariations thereof.

In another embodiment, the invention provides an isolated antimicrobialpeptide having an amino terminal amino acid sequence:

    NH.sub.2 -KKWWKX.                                          (SEQ ID NO:33)

Preferably, the peptide is from about 20 to about 30 amino acids inlength and therefore X is from about 14 to about 24 amino acids. Mostpreferably the peptide has a sequence of

                          (SEQ ID NO:20)                                          NH.sub.2 -KKWWKAQKAVNSGPNALQTLAQ-COOH                                                               (SEQ ID NO:21)                                          NH.sub.2 -KKWWKAKKFANSGPNALQTLAQ-COOH,                                        or                                                                                                  (SEQ ID NO:22)                                          NH.sub.2 -KKWWKFIKKAVNSGTTGLQTLAS-COOH,                                   

analogs, derivatives and conservative variations thereof, wherein thepeptides have antimicrobial activity. The peptides of the inventioninclude SEQ ID NO:20-22, as well as the broader groups of peptideshaving hydrophilic and hydrophobic substitutions, and conservativevariations thereof.

Other cationic peptides of the invention include:

                          (SEQ ID NO:23)                                          NH.sub.2 -KKSFFKKLTSVASSVLS-COOH (MBI 21A14),                                                       (SEQ ID NO:24)                                          NH.sub.2 -WKVFKSFIKKASSFAQSVLD-COOH,                                          and                                                                                                 (SEQ ID NO:25)                                          NH.sub.2 -KKWRKSFFKQVGSFDNSV-COOH,                                        

analogs, derivatives and conservative variations thereof, wherein thepeptides have antimicrobial activity.

The sequence of SEQ ID NO:1 contains helix-forming hydrophilic aminoacids at residues 4, 8, 10, 11, and 14. Amino acids 6 and 9 arehydrophobic residues (see FIG. 1). The helical nature of the first 10amino acids is depicted in FIGS. 1 and 2. The cationic charge of thepeptide of SEQ ID NO:1 was achieved by placing a lysine at positions 8and 14, thereby increasing the total positive charge to 6. The carboxyterminus of SEQ ID NO:1 was converted to the methyl ester and theantibacterial activity of this derivative was the same as that of theunmodified peptide. The peptide of SEQ ID NO:1 also has antifungalactivity. Amino acid residues 3, 4, 7, 8, 10, 11, 14, and 15 arepreferably hydrophilic residues, while amino acid residues 2, 5, 6, 9,12, and 13 are preferably hydrophobic residues.

SEQ ID NO:1 was modified to increase the alpha helical nature bychanging amino acids in the C-terminal tail, thereby resulting in SEQ IDNO:2. Amino acids 11 (neutral hydrophilic) and 13 (hydrophobic) of SEQID NO:1 were changed to another neutral hydrophilic and hydrophobicamino acid, respectively, in order to increase alpha helicity in thisregion. The C-terminal tail (residue 20-26) was modified to include apositive charge and increase alpha helicity. Therefore residues 20, 21,23, 24 and 25 were changed (See FIGS. 1 and 2). SEQ ID NO:3 was obtainedby changing amino acids 8, 11, 12, 13, 14, and 16 of SEQ ID NO:1. Thefirst 15 amino acid residues contain lysine at every fourth residue ofthe alpha helix. Thus, the lysines at positions 8 and 14 in SEQ ID NO:1were replaced by asparagine and serine, respectively, and the serine atposition 11 was changed to lysine. Further changes were made atpositions 12, 13 and 16 to conserve the helical nature. The C-terminaltail was unchanged. SEQ ID NO:4 is identical to SEQ ID NO:3, except forthe amino acid at position 4, which incorporates an additional positivecharge. SEQ ID NO:5 and SEQ ID NO:6 represent peptides having the sameamino acid sequence as SEQ ID NO:3 for the first 9 amino acids. Theamino acid at position 10 is glutamic acid. Residues 10, 11, 12, and 13in both SEQ ID NO:5 and SEQ ID NO:6 are different than SEQ ID NO:1, butremain either hydrophilic or hydrophobic as described above.

Residue 14 in SEQ ID NO:5 is the same as in SEQ ID NO:1. All residuesthereafter, 15-26, are completely changed to construct a hydrophobichelical tail, and bears no resemblance to the tail of SEQ ID NO:1. SEQID NO:6 contains the same residues as SEQ ID NO:1 at positions 14 and15. Residues 16-26 have been changed to form a helical tail.

The term "isolated" as used herein refers to a peptide substantiallyfree of proteins, lipids, nucleic acids, for example, with which it isnaturally associated. Those of skill in the art can make similarsubstitutions to achieve peptides with greater antibacterial activityand a broader host range. For example, the invention includes thebactolysin peptides depicted in SEQ ID NO:1-25, as well as analogues orderivatives thereof, as long as the bioactivity of the peptide remains.Minor modifications of the primary amino acid sequence of the peptidesof the invention may result in peptides which have substantiallyequivalent activity as compared to the specific peptides describedherein. Such modifications may be deliberate, as by site-directedmutagenesis, or may be spontaneous. All of the peptides produced bythese modifications are included herein as long as the biologicalactivity of the original peptide still exists.

Further, deletion of one or more amino acids can also result in amodification of the structure of the resultant molecule withoutsignificantly altering its biological activity. This can lead to thedevelopment of a smaller active molecule which would also have utility.For example, amino or carboxy terminal amino acids which may not berequired for biological activity of the particular peptide can beremoved. Peptides of the invention include any analog, homolog, mutant,isomer or derivative of the peptides disclosed in the present invention,so long as the bioactivity as described herein is remains. All peptideswere synthesized using L amino acids, however, all D forms of thepeptides (e.g., see Table 1b, CEMA) can be synthetically produced. Inaddition, C-terminal derivatives can be produced, such as C-terminalmethyl esters, in order to increase the antimicrobial activity of apeptide of the invention.

The peptide of the invention include peptides which are conservativevariations of those peptides specifically exemplified herein. The term"conservative variation" as used herein denotes the replacement of anamino acid residue by another, biologically similar residue. Examples ofconservative variations include the substitution of one hydrophobicresidue such as isoleucine, valine, leucine, alanine, cysteine, glycine,phenylalanine, proline, tryptophan, tyrosine, norleucine or methioninefor another, or the substitution of one polar residue for another, suchas the substitution of arginine for lysine, glutamic for aspartic acids,or glutamine for asparagine, and the like. Neutral hydrophilic aminoacids which can be substituted for one another include asparagine,glutamine, serine and threonine. The term "conservative variation" alsoincludes the use of a substituted amino acid in place of anunsubstituted parent amino acid provided that antibodies raised to thesubstituted polypeptide also immunoreact with the unsubstitutedpolypeptide. Such conservative substitutions are within the definitionof the classes of the peptides of the invention with respect to R₁ andR₂. For the peptides of the invention, a preferred conservativevariation is substitution of lysine by arginine.

The biological activity of the peptides can be determined by standardmethods known to those of skill in the art, such as "minimal inhibitoryconcentration (MIC)" assay described in the present examples, wherebythe lowest concentration at which no change in OD is observed for agiven period of time is recorded as MIC. Alternatively, "fractionalinhibitory concentration (FIC)" is also useful for determination ofsynergy between the peptides of the invention, or the peptides incombination with known antibiotics. FICs are performed by checkerboardtitrations of peptides in one dimension of a microtiter plate, and ofantibiotics in the other dimension, for example. The FIC is calculatedby looking at the impact of one antibiotic on the MIC of the other andvice versa. An FIC of one indicates that the influence of the compoundsis additive and an FIC of less than one indicates synergy. Preferably,an FIC of less than 0.5 is obtained for synergism. As used herein, FICcan be determined as follows: ##EQU1## Peptides of the invention can besynthesized by such commonly used methods as t-BOC or FMOC protection ofalpha-amino groups. Both methods involve stepwise syntheses whereby asingle amino acid is added at each step starting from the C terminus ofthe peptide (See, Coligan, et al, Current Protocols in Immunology, WileyInterscience, 1991, Unit 9). Peptides of the invention can also besynthesized by the well known solid phase peptide synthesis methodsdescribed Merrifield, J. Am. Chem. Soc., 85:2149, 1962), and Stewart andYoung, Solid Phase Peptides Synthesis, (Freeman, San Francisco, 1969,pp.27-62), using a copoly(styrene-divinylbenzene) containing 0.1-1.0mMol amines/g polymer. On completion of chemical synthesis, the peptidescan be deprotected and cleaved from the polymer by treatment with liquidHF-10% anisole for about 1/4-1 hours at 0° C. After evaporation of thereagents, the peptides are extracted from the polymer with 1% aceticacid solution which is then lyophilized to yield the crude material.This can normally be purified by such techniques as gel filtration onSephadex G-15 using 5% acetic acid as a solvent. Lyophilization ofappropriate fractions of the column will yield the homogeneous peptideor peptide derivatives, which can then be characterized by such standardtechniques as amino acid analysis, thin layer chromatography, highperformance liquid chromatography, ultraviolet absorption spectroscopy,molar rotation, solubility, and quantitated by the solid phase Edmandegradation.

The invention includes polynucleotides encoding:

                          (SEQ ID NO:26)                                          NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2          R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 VLTTGLPALIS-COOH,                                           (SEQ ID NO:27)                                          NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2          R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 VVTTAKPLISS-COOH,                                           (SEQ ID NO:28)                                          NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2          R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 ILTTGLPALIS-COOH,                                           (SEQ ID NO:29)                                          NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2          R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 GGLLSNIVTSL-COOH,                     and                                                                                                 (SEQ ID NO:30)                                          NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2          R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 GPILANLVSIV-COOH                  

wherein R₁ is a hydrophobic amino acid residue and R₂ is a hydrophilicamino acid residue. More specifically, the invention also includes anisolated polynucleotide which encodes the MBI 29 peptide of SEQ ID NO:1, an isolated polynucleotide which encodes the MBI 26 peptide of SEQ IDNO:2, and isolated polynucleotides which encode SEQ ID NO:3-25. Inaddition, the invention includes polynucleotides which encode analogs,mutants and variants of the peptides of the invention. The term"isolated" as used herein refers to a polynucleotide substantially freeof proteins, lipids, nucleic acids, for example, with which it isnaturally associated. As used herein, "polynucleotide" refers to apolymer of deoxyribonucleotides or ribonucleotides, in the form of aseparate fragment or as a component of a larger construct. DNA encodinga peptide of the invention can be assembled from cDNA fragments or fromoligonucleotides which provide a synthetic gene which is capable ofbeing expressed in a recombinant transcriptional unit. Polynucleotidesequences of the invention include DNA, RNA and cDNA sequences. Apolynucleotide sequence can be deduced from the genetic code, however,the degeneracy of the code must be taken into account. Polynucleotidesof the invention include sequences which are degenerate as a result ofthe genetic code. Such polynucleotides are useful for the recombinantproduction of large quantities of a peptide of interest, such as thepeptide of SEQ ID NO: 1-25.

In the present invention, the polynucleotides encoding the cationicpeptides of the invention may be inserted into a recombinant "expressionvector". The term "expression vector" refers to a plasmid, virus orother vehicle known in the art that has been manipulated by insertion orincorporation of cationic genetic sequences. Such expression vectors ofthe invention are preferably plasmids which contain a promoter sequencewhich facilitates the efficient transcription of the inserted geneticsequence in the host. The expression vector typically contains an originof replication, a promoter, as well as specific genes which allowphenotypic selection of the transformed cells. For example, theexpression of the peptides of the invention can be placed under controlof E. coli chromosomal DNA comprising a lactose or lac operon whichmediates lactose utilization by elaborating the enzymebeta-galactosidase. The lac control system can be induced by IPTG. Aplasmid can be constructed to contain the lac Iq repressor gene,permitting repression of the lac promoter until IPTG is added. Otherpromoter systems known in the art include beta lactamase, lambdapromoters, the protein A promoter, and the tryptophan promoter systems.While these are the most commonly used, other microbial promoters, bothinducible and constitutive, can be utilized as well. The vector containsa replicon site and control sequences which are derived from speciescompatible with the host cell. In addition, the vector may carryspecific gene(s) which are capable of providing phenotypic selection intransformed cells. For example, the beta-lactamase gene confersampicillin resistance to those transformed cells containing the vectorwith the beta-lactamase gene.

Transformation of a host cell with the polynucleotide may be carried outby conventional techniques well known to those skilled in the art. Forexample, where the host is prokaryotic, such as E. coli, competent cellswhich are capable of DNA uptake can be prepared from cells harvestedafter exponential growth and subsequently treated by the CaCl₂ methodusing procedures well known in the art. Alternatively, Mg₂ Cl or RbClcould be used.

In addition to conventional chemical methods of transformation, theplasmid vectors of the invention may be introduced into a host cell byphysical means, such as by electroporation or microinjection.Electroporation allows transfer of the vector by high voltage electricimpulse, which creates pores in the plasma membrane of the host and isperformed according to methods well known in the art. Additionally,cloned DNA can be introduced into host cells by protoplast fusion, usingmethods well known in the art.

DNA sequences encoding the cationic peptides can be expressed in vivo byDNA transfer into a suitable host cell. "Host cells" of the inventionare those in which a vector can be propagated and its DNA expressed. Theterm also includes any progeny of the subject host cell. It isunderstood that not all progeny are identical to the parental cell,since there may be mutations that occur during replication. However,such progeny are included when the terms above are used. Preferred hostcells of the invention include E. coli, S. aureus and P. aeruginosa,although other Gram-negative and Gram-positive organisms known in theart can be utilized as long as the expression vectors contain an originof replication to permit expression in the host.

The cationic peptide polynucleotide sequence used according to themethod of the invention can be isolated from an organism or synthesizedin the laboratory. Specific DNA sequences encoding the cationic peptideof interest can be obtained by: 1) isolation of a double-stranded DNAsequence from the genomic DNA; 2) chemical manufacture of a DNA sequenceto provide the necessary codons for the cationic peptide of interest;and 3) in vitro synthesis of a double-stranded DNA sequence by reversetranscription of mRNA isolated from a donor cell. In the latter case, adouble-stranded DNA complement of mRNA is eventually formed which isgenerally referred to as cDNA.

The synthesis of DNA sequences is frequently the method of choice whenthe entire sequence of amino acid residues of the desired peptideproduct is known. In the present invention, the synthesis of a DNAsequence has the advantage of allowing the incorporation of codons whichare more likely to be recognized by a bacterial host, thereby permittinghigh level expression without difficulties in translation. In addition,virtually any peptide can be synthesized, including those encodingnatural cationic peptides, variants of the same, or synthetic peptides.

When the entire sequence of the desired peptide is not known, the directsynthesis of DNA sequences is not possible and the method of choice isthe formation of cDNA sequences. Among the standard procedures forisolating cDNA sequences of interest is the formation of plasmid orphage containing cDNA libraries which are derived from reversetranscription of mRNA which is abundant in donor cells that have a highlevel of genetic expression. When used in combination with polymerasechain reaction technology, even rare expression products can be cloned.In those cases where significant portions of the amino acid sequence ofthe cationic peptide are known, the production of labeled single ordouble-stranded DNA or RNA probe sequences duplicating a sequenceputatively present in the target cDNA may be employed in DNA/DNAhybridization procedures which are carried out on cloned copies of thecDNA which have been denatured into a single stranded form (Jay, et al.,Nuc. Acid Res., 11:2325, 1983).

The invention also provides a method of inhibiting the growth ofbacteria comprising contacting the bacteria with an inhibiting effectiveamount of a peptide of the invention, including:

                          (SEQ ID NO:26)                                          NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2          R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 VLTTGLPALIS-COOH,                                           (SEQ ID NO:27)                                          NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2          R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 VVTTAKPLISS-COOH,                                           (SEQ ID NO:28)                                          NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2          R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 ILTTGLPALIS-COOH,                                           (SEQ ID NO:29)                                          NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2          R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 GGLLSNIVTSL-COOH,                                           (SEQ ID NO:30)                                          NH.sub.2 -KWKR.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 R.sub.1 R.sub.2          R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2 GPILANLVSIV-COOH,                                           (SEQ ID NO:31)                                          NH.sub.2 -KKWWRRR.sub.1 R.sub.1 R.sub.2 R.sub.1 R.sub.1 R.sub.2 R.sub.2       GPALSNV-COOH,                                                                 or                                                                                                  (SEQ ID NO:35)                                          NH.sub.2 -KKWWRRR.sub.1 R.sub.1 R.sub.2 GLKTAGPAIQSVLNK-COOH              

wherein R₁ is a hydrophobic amino acid residue and R₂ is a hydrophilicamino acid residue. More specifically, the invention provides a methodof inhibiting the growth of bacteria comprising contacting the bacteriawith an inhibiting effective amount of a peptide of the invention, suchas MBI 26 or MBI 29,

                          (SEQ ID NO:1)                                           NH.sub.2 -KWKSFIKKLTTAVKKVLTTGLPALIS-COOH,                                                          (SEQ ID NO:2)                                           NH.sub.2 -KWKSFIKKLTSAAKKVVTTAKPLISS-COOH,                                    or                                                                                                  (SEQ ID NO:3)                                           NH.sub.2 -KWKSFIKNLTKGGSKILTTGLPALIS-COOH,                                                          (SEQ ID NO:4)                                           NH.sub.2 -KWKKFIKNLTKGGSKILTTGLPALIS-COOH,                                                          (SEQ ID NO:5)                                           NH.sub.2 -KWKSFIKNLEKVLKPGGLLSNIVTSL-COOH                                     (MBI 490),                                                                                          (SEQ ID NO:6)                                           NH.sub.2 -KWKSFIKNLEKVLKKGPILANLVSIV-COOH                                     (MBI 491),                                                                                          (SEQ ID NO:7)                                           NH.sub.2 -KWKEFIKKLTTAVKKVLTTGLPALIS-COOH                                     (MBI 492),                                                                                          (SEQ ID NO:8)                                           NH.sub.2 -KWKKFIKELQKVLAPGGLLSNIVTSL-COOH                                     (MBI 493),                                                                                          (SEQ ID NO:9)                                           NH.sub.2 -KWKSFIKKLTSVLKKVVTTALPALIS-COOH                                     (MBI 29A1),                                                                                         (SEQ ID NO:10)                                          NH.sub.2 -KWKSFIKNLTKVLKKVVTTALPALIS-COOH                                     (MBI 29A2),                                                                                         (SEQ ID NO:11)                                          NH.sub.2 -KWKLFKKKGTGAVLTVLTTGLPALIS-COOH                                     (MBI 29A3),                                                                                         (SEQ ID NO:12)                                          NH.sub.2 -KWKSFIKKLTSVLKKVVTTAKPLISS-COOH,                                                          (SEQ ID NO:13)                                          NH.sub.2 -KKKSFIKLLTSAKVSVLTTAKPLISS-COOH,                                                          (SEQ ID NO:14)                                          NH.sub.2 -KWKKFIKELQKVLKPGGLLSNIVTSL-COOH,                                                          (SEQ ID NO:15)                                          NH.sub.2 -KKWWRRVLSGLKTGPALSNV-COOH,                                                                (SEQ ID NO:16)                                          NH.sub.2 -KKWWRRVLKGLSSGPALSNV-COOH,                                                                (SEQ ID NO:17)                                          NH.sub.2 -KKWWRRALQALKNGPALSNV-COOH,                                                                (SEQ ID NO:18)                                          NH.sub.2 -KKWWRRVLSGLKTAGPAIQSVLNK-COOH                                       (MBI 21A1),                                                                                         (SEQ ID NO:19)                                          NH.sub.2 -KKWWRRALQGLKTAGPAIQSVLNK-COOH                                       (MBI 21A2),                                                                                         (SEQ ID NO:20)                                          NH.sub.2 -KKWWKAQKAVNSGPNALQTLAQ-COOH                                                               (SEQ ID NO:21)                                          NH.sub.2 -KKWWKAKKFANSGPNALQTLAQ-COOH,                                                              (SEQ ID NO:22)                                          NH.sub.2 -KKWWKFIKKAVNSGTTGLQTLAS-COOH,                                                             (SEQ ID NO:23)                                          NH.sub.2 -KKSFFKKLTSVASSVLS-COOH                                              (MBI 21A14),                                                                                        (SEQ ID NO:24)                                          NH.sub.2 -WKVFKSFIKKASSFAQSVLD-COOH,                                          and                                                                                                 (SEQ ID NO:25)                                          NH.sub.2 -KKWRKSFFKQVGSFDNSV-COOH.                                        

and analogs, derivatives, or conservative variations thereof. The term"contacting" refers to exposing the bacteria to the peptide so that thepeptide can effectively inhibit, kill, or lyse bacteria, bind endotoxin(LPS), or permeabilize gram-negative bacterial outer membranes.Contacting may be in vitro, for example by adding the peptide to abacterial culture to test for susceptibility of the bacteria to thepeptide. Contacting may be in vivo, for example administering thepeptide to a subject with a bacterial disorder, such as septic shock."Inhibiting" or "inhibiting effective amount" refers to the amount ofpeptide which is required to cause a bacteriostatic or bactericidaleffect. Examples of bacteria which may be inhibited include E. coli, P.aeruginosa, E. cloacae, S. typhimurium, and S. aureus.

The method of inhibiting the growth of bacteria may further include theaddition of antibiotics for combination or synergistic therapy. Theappropriate antibiotic administered will typically depend on thesusceptibility of the bacteria such as whether the bacteria is gramnegative or gram positive, and will be easily discernable by one ofskill in the art. Examples of particular classes of antibiotics usefulfor synergistic therapy with the peptides of the invention includeaminoglycosides (e.g., tobramycin), penicillins (e.g., piperacillin),cephalosporins (e.g., ceftazidime), fluoroquinolones (e.g.,ciprofloxacin), carbepenems (e.g., imipenem), tetracyclines andmacrolides (e.g., erythromycin and clarithromycin). The peptides and/oranalogues or derivatives thereof may be administered to any host,including a human or non-human animal, in an amount effective to inhibitnot only growth of a bacterium, but also a virus or fungus. Thesepeptides are useful as antimicrobial agents, antiviral agents, andantifungal agents.

The peptide of the invention can be administered parenterally byinjection or by gradual infusion over time. The peptide can beadministered intravenously, intraperitoneally, intramuscularly,subcutaneously, intracavity, or transdermally. Preferred methods fordelivery of the peptide include orally, by encapsulation in microspheresor proteinoids, by aerosol delivery to the lungs, or transdermally byiontophoresis or transdermal electroporation. Other methods ofadministration will be known to those skilled in the art.

Preparations for parenteral administration of a peptide of the inventioninclude sterile aqueous or non-aqueous solutions, suspensions, andemulsions. Examples of non-aqueous solvents are propylene glycol,polyethylene glycol, vegetable oils such as olive oil, and injectableorganic esters such as ethyl oleate. Aqueous carriers include water,alcoholic/aqueous solutions, emulsions or suspensions, including salineand buffered media. Parenteral vehicles include sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's, or fixed oils. Intravenous vehicles include fluid and nutrientreplenishers, electrolyte replenishers (such as those based on Ringer'sdextrose), and the like. Preservatives and other additives may also bepresent such as, for example, antimicrobials, anti-oxidants, chelatingagents, and inert gases and the like.

The invention provides a method of treating or ameliorating anendotoxemia or septic shock (sepsis) associated disorder, or one or moreof the symptoms of sepsis comprising administering to a subjectdisplaying symptoms of sepsis or at risk for developing sepsis, atherapeutically effective amount of a cationic peptide of the invention,for example, SEQ ID NO:1 or SEQ ID NO:2, or analogs, derivatives, orconservative variations thereof. The term "ameliorate" refers to adecrease or lessening of the symptoms of the disorder being treated.Such symptoms which may be ameliorated include those associated with atransient increase in the blood level of TNF, such as fever,hypotension, neutropenia, leukopenia, thrombocytopenia, disseminatedintravascular coagulation, adult respiratory distress syndrome, shockand multiple organ failure. Patients who require such treatment includethose at risk for or those suffering from toxemia, such as endotoxemiaresulting from a gram-negative bacterial infection, venom poisoning, orhepatic failure, for example. In addition, patients having agram-positive bacterial, viral or fungal infection may display symptomsof sepsis and may benefit from such a therapeutic method as describedherein. Those patients who are more particularly able to benefit fromthe method of the invention are those suffering from infection by E.coli, Haemophilus influenza B, Neisseria meningitides, staphylococci, orpneumococci. Patients at risk for sepsis include those suffering fromgunshot wounds, renal or hepatic failure, trauma, burns,immunocompromised (HIV), hematopoietic neoplasias, multiple myeloma,Castleman's disease or cardiac myxoma.

The term "therapeutically effective amount" as used herein for treatmentof endotoxemia refers to the amount of cationic peptide used is ofsufficient quantity to decrease the subject's response to LPS anddecrease the symptoms of sepsis. The term "therapeutically effective"therefore includes that the amount of cationic peptide sufficient toprevent, and preferably reduce by at least 50%, and more preferablysufficient to reduce by 90%, a clinically significant increase in theplasma level of TNF. The dosage ranges for the administration ofcationic peptide are those large enough to produce the desired effect.Generally, the dosage will vary with the age, condition, sex, and extentof the infection with bacteria or other agent as described above, in thepatient and can be determined by one skilled in the art. The dosage canbe adjusted by the individual physician in the event of anycontraindications. In any event, the effectiveness of treatment can bedetermined by monitoring the level of LPS and TNF in a patient. Adecrease in serum LPS and TNF levels should correlate with recovery ofthe patient.

In addition, patients at risk for or exhibiting the symptoms of sepsiscan be treated by the method as described above, further comprisingadministering, substantially simultaneously with the therapeuticadministration of cationic peptide, an inhibitor of TNF, an antibiotic,or both. For example, intervention in the role of TNF in sepsis, eitherdirectly or indirectly, such as by use of an anti-TNF antibody and/or aTNF antagonist, can prevent or ameliorate the symptoms of sepsis.Particularly preferred is the use of an anti-TNF antibody as an activeingredient, such as a monoclonal antibody with TNF specificity asdescribed by Tracey, et al. (Nature, 330:662, 1987).

A patient who exhibits the symptoms of sepsis may be treated with anantibiotic in addition to the treatment with cationic peptide. Typicalantibiotics include an aminoglycoside, such as gentamicin or abeta-lactam such as penicillin, or cephalosporin. Therefore, a preferredtherapeutic method of the invention includes administering atherapeutically effective amount of cationic peptide substantiallysimultaneously with administration of a bactericidal amount of anantibiotic. Preferably, administration of cationic peptide occurs withinabout 48 hours and preferably within about 2-8 hours, and mostpreferably, substantially concurrently with administration of theantibiotic.

The term "bactericidal amount" as used herein refers to an amountsufficient to achieve a bacteria-killing blood concentration in thepatient receiving the treatment. The bactericidal amount of antibioticgenerally recognized as safe for administration to a human is well knownin the art, and as is known in the art, varies with the specificantibiotic and the type of bacterial infection being treated.

Because of the antibiotic, antimicrobial, and antiviral properties ofthe peptides, they may also be used as preservatives or sterillants ofmaterials susceptible to microbial or viral contamination. The peptidesof the invention can be utilized as broad spectrum antimicrobial agentsdirected toward various specific applications. Such applications includeuse of the peptides as preservatives in processed foods (organismsincluding Salmonella, Yersinia, Shigella), either alone or incombination with antibacterial food additives such as lysozymes; as atopical agent (Pseudomonas, Streptococcus) and to kill odor producingmicrobes (Micrococci). The relative effectiveness of the cationicpeptides of the invention for the applications described can be readilydetermined by one of skill in the art by determining the sensitivity ofany organism to one of the peptides.

The following examples are intended to illustrate but not limit theinvention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may alternatively be used.

EXAMPLE 1 MIC VALUES FOR CATIONIC PEPTIDES

The minimum inhibitory concentrations of (MIC) of CEME, CEMA, the first8 amino acid residues of

    CEME (NH.sub.2 -KWKLFKKIGIGAVLKVLTTGLPALIS-COOH; SEQ ID NO:1

with changes at residues 4, 6, 8-11, 14) and

    CEMA (NH.sub.2 -KWKLFKKIGIGAVLKVLTTGLPALKLTK-COOH; SEQ NO:1

with changes at residues 4, 6, 8-11, 14, and 25-28), (Piers, K. andHancock, R, Molec. Microbiology, 12(6), 1994) 20 carboxy terminal aminoacids of melittin (MA), 8 amino terminal amino acids from cecropin, andthe peptides shown in SEQ ID NO:1, MBI 29 and SEQ ID NO:2, MBI 26, weredetermined for a number of different bacteria (Table 1a and 1b).Briefly, cells were grown overnight at 37° C. in LB-S (Luria brothwithout any salt supplement) and diluted one in 10,000 in the samemedium to give concentrations of about 10⁴ to 10⁵ CFU/ml. The brothdilutions were set up in a 96 well microtiter plate by putting 200 μl ofLB-S containing the initial concentration of antibiotic or compound incolumn 1 and 100 μl of the same medium in columns 2-12. The compoundswere diluted by taking 100 μl of broth from column 1 and mixing it withcolumn 2, resulting in a one in two dilution. This was continued tocolumn 10. Finally, 10 μl of bacteria were pipetted into columns 1-11,and the plates incubated overnight at 37° C. The next day the plateswere scored for growth in the wells, and the MIC determined.

                                      TABLE 1a                                    __________________________________________________________________________    MINIMUM INHIBITORY CONCENTRATION                                              (MIC) VALUES FOR CATIONIC PEPTIDES                                            __________________________________________________________________________    STRAIN   CEME                                                                              CEMA  MBI 29                                                                            MBI 26                                                                             CE-8                                                                             MA-20                                          __________________________________________________________________________    S. aureus                                                                              32  ≧64                                                                          20  ≧64                                                                         ≧64                                                                       ≧64                                     (K147)                                                                        S. aureus                                                                              24  ≧64                                                                          18  ≧64                                                                         ≧64                                                                       ≧64                                     (SAP0017)                                                                     S. epidermis                                                                           16  ≧64                                                                          10  ≧64                                                                         ≧64                                                                       ≧64                                     E. coli  5   10    3   3    ≧64                                                                       ≧64                                     (UB1005)                                                                      P. aeruginosa                                                                          8   26    5   11   ≧64                                                                       ≧64                                     (H187)                                                                        Candida albicans                                                                       ≧64                                                                        ≧64                                                                          40  ≧64                                                                         ≧64                                                                       ≧64                                     (CAN105)                                                                      __________________________________________________________________________    Additional MICs were determined for MBI-26 and MBI-29 and MBI 29              and MBI21 derivatives as noted for the following strains:                     Strain         MBI 26                                                                            MBI 29                                                     __________________________________________________________________________    P. aeruginosa  4   4                                                          (H103)                                                                        E. coli. Bort  2   6                                                          E. coli 0111:B4                                                                              2   2                                                          X maltophilia  2   3                                                          A. calcoaceticus                                                                             2   4                                                          E. cloacae     2   2                                                          K. pneumoniae  8   16                                                         __________________________________________________________________________          E. coli                                                                       0111:B4           S. epi                                                Peptide                                                                             μg/ml                                                                           E. faecalis                                                                        B. Subtilis                                                                        H103                                                                             0017                                                                             SAP                                                                              K147                                                                             C610                                         __________________________________________________________________________    MBI29A1                                                                             4    64   16   16 16 16 16 8                                            MBI29A2                                                                             8    64   8    16 16 8  16 8                                            MBI29A3                                                                             8    >128 16   16 16 32 32 8                                            MBI21A1                                                                             16   >128 >128 64 128                                                                              >28                                                                              >128                                                                             16                                           MBI21A2                                                                             32   >128 >28  128                                                                              >128                                                                             >128                                                                             >128                                                                             16                                           MBI21A14                                                                            >128 >128 >128 >128                                                                             >128                                                                             >128                                                                             >128                                                                             128                                          __________________________________________________________________________

In a separate set of experiments, the following MIC values wereobtained:

                                      TABLE lb                                    __________________________________________________________________________    MINIMUM INHIBITORY CONCENTRATION                                              (MIC) VALUES FOR CATIONIC PEPTIDES                                            STRAIN  CEMA                                                                              CEMA.sup.me                                                                        MBI 29                                                                            MBI 29.sup.me                                                                      MBI 26D                                                                            MBI 26L                                        __________________________________________________________________________    S. aureus                                                                             ≧64                                                                        ≧64                                                                         32  16   ≧64                                                                         ≧64                                     (K147)                                                                        S. aureus                                                                             ≧64                                                                        ≧64                                                                         32  16   ≧64                                                                         ≧64                                     (SAP0017)                                                                     S. epidermis                                                                          43  43   16  8    8-16 32                                             P. aeruginosa                                                                         32  53   16  8    16   32                                             (H187)                                                                        E. coli 13  32   4   4    4-8  4                                              (UB1005)                                                                      E. faecalis                                                                           ≧64                                                                        ≧64                                                                         32  ≧64                                                                         ≧64                                                                         ≧64                                     Candida albicans                                                                      ≧64                                                                        ≧64                                                                         32  32   ≧64                                                                         ≧64                                     (CAN105)                                                                      __________________________________________________________________________

The results show that both MBI 29 (SEQ ID NO:1) and MBI 26 (SEQ ID NO:2)are effective antimicrobial agents for a variety of gram positive andgram negative bacteria. In addition, all peptides were effective againstCandida albicans. Modification of the peptides, such as methyl estermodification, or L to D amino acids provided a broader class ofantimicrobial agents.

Agarose Dilution Susceptibility Testing

The following is a novel agarose-based solid phase assay system tomeasure antimicrobial activity of the cationic peptides of theinvention, for example, either alone or in combination with aconventional antibiotic. Standard MIC methods, as described above orthis method can be used to determine MICs of the peptides describedherein.

A) Preparing plates

1) Unsupplemented Mueller-Hinton agarose (i.e. no Ca²⁺ or Mg²⁺ added)was used. (Unsupplemented broth is used with agar because agar containsdivalent cations).

Prepared as follows:

22 g/L Mueller-Hinton powder (BBI, #11443)

15 g/L agarose (Sigma, A-0576)

QS to 1 L with sdH₂ O

Heat to boiling and boil 1 min

Adjust pH to 7.3 with NaOH or KOH (adjust the meter to the correcttemperature--note agarose solidifies at <40° C.)

Autoclave for 10 min at 121° C.

After the agarose has been autoclaved, it is allowed to cool to 40-45°C. in a water bath before aseptically adding antimicrobial solutions,and pouring the plates.

2) Appropriate dilutions of antimicrobial solutions are added to moltentest agarose that has been allowed to equilibrate in a water bath to40-45° C. In general, a scheme in which 1 part of antimicrobial solutionis added to 9 parts of liquid agarose is used (i.e. 2 mL of drug to 18mL of agar for each plate; 1 mL of each drug in the combination iftesting synergy).

3) The agarose and antimicrobial solution are mixed throughly by gentleinversion about 10× to avoid bubbles and the mixture poured intopetri-dishes on a level surface to result in an agar depth of 3-4 mm.

4) The plates are poured as quickly as possible after mixing to preventcooling and partial solidification.

5) The agarose is allowed to solidify at room temperature and the platesare either used immediately or stored in sealed plastic bags at 4° C.Storage time can vary but plates should be used within a week.

6) Plates stored at 4° C. should be allowed to equilibrate at roomtemperature before use. The surface should be dry before inoculating theplates. If necessary, plates can be placed in the fume hood for 30 minwith lids ajar to hasten drying of agarose surface.

B) Inoculum Preparation

1) To standardize the inoculum density, a 0.5 McFarland standard can beprepared as follows:

A 0.5 mL aliquot of 0.48 m BaCl₂ is added to 99.5 mL of 0.36 N H₂ SO₄(1% v/v) with constant stirring to maintain a suspension. The correctdensity should be verified using a spectrophotometer with a 1 cm lightpath and matched cuvettes to determine the absorbance. The absorbance at630 nm should be 0.08 to 0.10. The suspension should be transferred to10 mL aliquot into screwcap tubes of the same size as those used ingrowing or diluting the bacterial inoculum. These tubes should betightly sealed and stored in the dark at room temperature. The turbiditystandard should be rigorously agitated on a vortex mixer prior to use.If large particles appear, the standard should be replaced. Replacementshould occur every month (use a new standard tubes each time, and whenten are used up, make a new batch).

2) 3 to 5 morphologically identical colonies are selected from a TSAplate (the top of each is touched with a sterile loop) and transferredto a tube of 5 mL TSB.

3) The broth culture is incubated at 37° C. with shaking until itachieved or exceeds the turbidity of the standard (2 to 6hours)--approx. 1 to 2×10⁸ CFU/mL.

4) The turbidity can be adjusted with broth to obtain a turbidity equalto the 0.5 standard. Use adequate lighting and hold up a white card withblack lines behind the cultures to assist in the comparison.

5) Cultures adjusted to the standard are diluted 1/10 in sterile brothto obtain a density of approximately 10⁷ CFU/mL. Most inoculumreplicators deposit approximately 1 to 2 μL on the agarose surface. Thefinal inoculum on the agarose will then be approximately 10⁴ CFU in aspot of 5 to 8 mm in diameter on the agarose. The adjusted suspensionsshould be used for final inoculation within 15 min of preparation.

The tubes containing the adjusted and diluted bacterial suspensionsshould be arranged in order on a rack. An aliquot of each well-mixedsuspension is placed into the corresponding well in the replicatorinoculum block. The agarose plates are marked for orientation of theinoculum spots. A growth control plate (no antimicrobial) is inoculatedfirst and then, starting with the lowest concentration, the platescontinuing the different antimicrobial concentration are inoculated(Optional--A second growth control plate is inoculated last to ensurethat there was no contamination or significant antimicrobial carryoverduring the inoculation).

Optional--a sample of each inoculum is streaked on a suitable agar plateand incubated

6) The inoculated plates are allowed to stand at room temperature untilthe moisture in the inoculum spots have been absorbed into the agarose.The plates are inverted and incubated at 35° C. for 24 hours, 48 hours.

7) The plates should be placed on a dark, nonreflecting surface todetermine the end points. The MIC is recorded as the lowestconcentration of antimicrobial agent that completely inhibits growth,disregarding a single-colony or a faint haze caused by the inoculum.

8) If two or more colonies persist in concentrations of the agent beyondan obvious end point, or if there is no growth at lower concentrations,the culture purity is checked and test possibly repeated.

9) After 48 hours a replica plating tool can be used to transfer cellsonto a fresh TSA or BHI plate and growth is read after 24 hrs.

EXAMPLE 2 EFFECT OF FORMULATION ON BACTERICIDAL ACTIVITY

Effect of formulation on bactericidal activity and MIC was determinedusing the test solutions listed below. The results show that citrate andrelated formulations reduced the MIC (Table 2). Similar effects wereseen with acetate formulated peptide (Tables 2 and 3).

                  TABLE 2                                                         ______________________________________                                        EFFECT OF CITRATE, ASCORBIC ACID AND DEXTROSE ON                              MBI 26 (10 MG/ML) KILLING OF P. AERUGINOSA                                               Survivors at Time (min.)                                           Test Solution                                                                         Peptide  0        15     30     60                                    ______________________________________                                        Citrate Control  1.1 × 10.sup.7                                                                   1.1 × 10.sup.7                                                                 1.2 × 10.sup.7                                                                 1.4 × 10.sup.7                  (0.129M)                                                                              + MBI 26 1.1 × 10.sup.7                                                                   0      0      0                                     Citrate Control  1.3 × 10.sup.7                                                                   1.2 × 10.sup.7                                                                 1.7 × 10.sup.7                                                                 1.7 × 10.sup.7                  (0.129M)                                                                              + MBI 26 1.3 × 10.sup.7                                                                   0      0      0                                     + Ascorbic                                                                    acid                                                                          (100 mM)                                                                      Citrate-                                                                              Control  1.2 × 10.sup.7                                                                   5.8 × 10.sup.6                                                                        6.9 × 10.sup.6                  Dextrose                                                                              + MBI 26 9 × 10.sup.6                                                                     20     0      0                                     Citrate-                                                                              Control  7.8 × 10.sup.6                                                                   6.5 × 10.sup.7                                                                 1.5 × 10.sup.7                                                                 1.1 × 10.sup.7                  Phosphate                                                                     Dextrose                                                                              + MBI 26 0               0      0                                     Dextran Control  2.3 × 10.sup.7                                                                   2.2 × 10.sup.7                                                                 1.4 × 10.sup.7                                                                 2 × 10.sup.7                            + MBI 26 1.9 × 10.sup.7                                                                   1.2 × 10.sup.6                                                                 1 × 10.sup.6                                                                   8.5 × 10.sup.5                          (20 μg)                                                            ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        EFFECT OF CITRATE ADDITION TO CATION ADJUSTED                                 MEDIA ON MINIMAL INHIBITORY CONCENTRATIONS                                                   MIC (μg/mL)                                                 Strain         CAMHB      CAMHB + Citrate                                     ______________________________________                                        E. coli (UB 1005)                                                                            4          2                                                   E. coli (111: B4)                                                                            8          2                                                   E. coli (Bort) 16         8                                                   S. typhimurium (C610)                                                                        8          2                                                   E. facaelis (C625)                                                                           16         >32                                                 A. calcoacet. (C402)                                                                         8          4                                                   A. calcoacet (C403)                                                                          1          1                                                   E. cloacae (C601)                                                                            8          4                                                   E. cloacae (C602)                                                                            8          4                                                   E. cloacae (C603)                                                                            4          2                                                   P. aeruginosa (H103)                                                                         16         2                                                   ______________________________________                                         The media used in this experiment was Cation Adjusted Muller Hinton           (CAMHB), and CAMHB + 8 mM citrate. MBI 26 was prepared in sterile             distilled water.                                                         

EXAMPLE 3 INNER MEMBRANE PERMEABILIZATION

Inner membrane permeability was determined by measurement in E. coli ML35 of β-galactosidase activity using ONPG as a substrate. Normally ONPGcannot pass across the cytoplasmic membrane to permit hydrolysis by thecytoplasmic enzyme β-galactosidase. However, cationic peptides, bypermeabilizing the inner membrane, unmasks this enzyme leading to colordevelopment as ONP is released. MBI 26 and MBI 29 were tested with thesame conditions as shown in FIG. 3 and 4 (4 ug MBI 26 or MBI 29 alone,; +100mM NaCl, □; +100 uM CCCP, ; +5 mM Mg, .sup.▪▪ ). While 100 mMNaCl had little effect on MBI 26 permeabilization, 5 mM Mg++ had a largeinhibitory effect. MBI 29 permeabilization of the inner membrane was theleast affected by any of the conditions especially Mg⁺⁺ as seen in FIG.4. Addition of citrate increased the lag time before the peptides wereable to permeabilize the membranes. When the experiments were executedin cation adjusted media (CAMHB), the lag time was increased up to 30minutes. The data in Table 4 shows the rate and lag time for innermembrane permeabilization of MBI 26, MBI 29 and peptides 490, 491, 492and 493 (SEQ ID NO:2, 1, and 5-8, respectively).

                  TABLE 4                                                         ______________________________________                                        INNER MEMBRANE PERMEABILIZIATION                                                                        Rate of Hydrolysis                                  Peptide       Lag Time (Sec.)                                                                           (ΔAU min.sup.-1)                              ______________________________________                                        MBI 26 (SEQ ID NO:2)                                                                        210         0.081                                               MBI 29 (SEQ ID NO:1)                                                                         66         0.21                                                490 (SEQ ID NO:5)                                                                           156         0.081                                               491 (SEQ ID NO:6)                                                                           152         0.14                                                492 (SEQ ID NO:7)                                                                            68         0.23                                                493 (SEQ ID NO:8)                                                                           136         0.14                                                ______________________________________                                    

EXAMPLE 4 INHIBITION OF LPS-MEDIATED TNF INDUCTION IN MACROPHAGES BYCATIONIC PEPTIDES

The effect of cationic peptides,

    CEME (KWKLFKKIGIGAVLKVLTTGLPALIS                           (SEQ ID NO:36))

and

    CEMA (KWKLFKKIGIGAVLKVLTTGLPALKLTK                         (SEQ ID NO:37))

and the peptides of the invention, MBI 29,

    NH.sub.2 -KWKSFIKKLTTAVKKVLTTGLPALIS-COOH                  (SEQ ID NO:1)

and MBI 26,

    NH.sub.2 -KWKSFIKKLTSAAKKVVTTAKPLISS-COOH                  (SEQ ID NO:2),

on LPS-induced TNF in macrophages was examined. RAW 264.7 macrophagecells were grown by seeding 10⁶ cells into a 162 cm² cell culture flaskand incubated at 37° C., 5% CO₂ for 1 week. RAW cell media [(Dulbecco'sModified Eagle Medium with Hepes buffer 450 ml; 2.4 mM L-glutamine 3 ml(400 mM); Pen/Strep 3 ml (10⁴ U/ml of Pen, 1 mg/ml strep); and 10% heatinactivated fetal bovine serum (FBS) 50 ml)] was then completely removedfrom flasks. 10 mls of cell dissociation solution (Sigma) was added toeach flask and incubated at 37° C. for 10 minutes. Cells were removedfrom flasks, diluted in RAW cell media and centrifuged for 6 minutes.The cell pellet was resuspended in 5 ml of media/flask used. 100 μl cellsuspension was removed and added to 400 μl of trypan blue and cells werecounted using a hemocytometer. The cell suspension was diluted to 1×10⁶cells/ml and 1 ml of suspension was added per well of a 24 well plate.The 24 well plates were incubated at 37° C., 5% CO₂ overnight for use inthe assay.

After an overnight incubation, the media was aspirated from all thewells. 100 μl of Lipopolysaccharide (LPS) was added at 100 ng/100 μl.CEME, CEMA or MBI 29 was added at the desired concentration/100 μl tospecified wells. RAW cell media was added to all the wells so they allhad a final volume of 1 ml. The plates were then incubated for six hoursat 37° C., 5% CO₂. The supernatant was then removed from the wells andstored overnight at 4° C. For those wells in which whole bacteria wereadded directly to the wells, the supernatant was centrifuged in 0.2 μmfilter eppendorf tubes for 5 minutes.

The supernatants were then used in cell cytotoxic L929 assay. Thesamples were transferred to 96 well plates. 50 μl of TNF media was addedto all the wells in all the plates except to those wells in the firstrow. 10 μl of murine TNF standard (20 ng/ml) and 90 μl of TNF media wasadded in duplicate to the plate and diluted 1:2 down the plate to thesecond to last row. Test samples (75 μl), comprising the supernatantsfrom the RAW cell assays, were added to separate rows in duplicate anddiluted 1:3 to the second to last rows.

TNF-sensitive L929 mouse fibroblast cells were grown by seeding 10⁶cells into a 162 cm² cell culture flask and left to grow for 1 week.L929 cells were removed from the flask with 10 mls of trypsin-EDTA/flaskand incubated 3-5 minutes. Cell suspension was diluted and centrifugedfor 6 minutes. The pellet was resuspended in 5 mls of fresh L929media/flask and counted (same as RAW cells). Cell suspension was dilutedto 10⁶ cells/ml. 100 μl was used to inoculate each well of the 96 wellplates with the supernatants. (L929 Growth Media was the same as RAWcell media except instead of FBS, 50 mls of 10% heat inactivated horseserum was utilized; TNF Assay Media was the same as RAW cell mediaexcept 4 μg/ml Actinomycin D was added.)

The plates were incubated at 37° C. at 5% CO₂ for 2 days. The media wasthen aspirated and replaced with 100 μl of the dye MTT (0.5 mg/ml) inmodified Eagle Medium without phenol red. The plates were then incubatedat 37° C. at 5% CO₂ for 3 hours. The dye was then removed and replacedwith 100 μl of absolute ethanol. The plates were left at roomtemperature for 10-15 minutes to dissolve the formazan dye crystals.

The plates were read at 570 nm in a ELISA plate reader with 690 nmreference filter. One unit of TNF activity is defined as the amountrequired to kill 50% of the L929 cells. The TNF level in Units per mltherefore was the reciprocal of the dilution which led to a 50% killingof L929 cells.

FIGS. 5 and 6 show levels of TNF (U/ml) after a 6 hour treatment withincreasing amounts (0, 5, 10, 20, 30, 40 or 50 μg) of either CEME (ME),CEMA (MA), MBI 26 (SEQ ID NO:2) or MBI 29 (SEQ ID NO:1) peptide and 100ng of LPS. TNF levels were measured six hours after the addition of E.coli 0111:B4 or E. coli Bort LPS (FIG. 5a, 5b, 5c and FIG. 6a and 6c aslabeled). P. aeruginosa LPS was also added in FIG. 6b. The data showsthe results of several separate experiments and indicate that bothpeptides efficiently reduce the level of LPS-induced TNF in the culturewith three distinct LPS samples at concentrations of peptides as low as5 μg/ml.

In addition, the peptides of SEQ ID NO: 5-8 also bound effectively toLPS with high affinity based on Dansyl polymyxin displacement assays(Table 5).

                  TABLE 5                                                         ______________________________________                                        Peptide            I.sub.50 (mM)                                                                          I.sub.max (%)                                     ______________________________________                                        MBI 26 (SEQ ID NO:2)                                                                             1.8      38                                                MBI 29 (SEQ ID NO:1)                                                                             2.8      40                                                490 (SEQ ID NO:5)  4.9      40                                                491 (SEQ ID NO:6)  4.5      24                                                492 (SEQ ID NO:7)  5.4      48                                                493 (SEQ ID NO:8)  3.8      56                                                ______________________________________                                    

EXAMPLE 5 CATIONIC PEPTIDE REDUCTION OF LPS-INDUCED TNF

In order to determine how rapidly the cationic peptides reducedLPS-induced TNF production, E. coli 0111:B4 was added at time 0 to RAWmacrophages. MBI 29 or Polymyxin B was added at time 0, 30 and 60minutes. Levels of TNF were measured after 6 hours. The results areshown in FIG. 7a and 7b(PMB, Polymyxin B; 29, MBI 29 (SEQ ID NO:1)). Theresults show that MBI 29 inhibited TNF induction by LPS in a similarmanner to polymyxin B. Furthermore, MBI 29 was effective at reducing theability of LPS to induce TNF in RAW cell lines even when added 60minutes after the addition of LPS. MBI 29 demonstrated a distinct andreproducible advantage over polymyxin B when added 60 minutes after theaddition of LPS. To confirm that MBI 29 was acting on LPS rather thandirectly upon macrophage cell lines, 20 μg of MBI 29 was added to RAWcells and incubated for 60 minutes prior to aspiration of the medium andwashing the cells 3 times with HBSS (Hanks Buffered Salt Solution).Addition of 10 ng or 100 ng of LPS to the washed RAW cells resulted in ahigh level of TNF induction (14,000-20,000 Units of TNF per ml),suggesting that the MBI 29 had not permanently depressed the ability ofRAW cells to induce TNF in response to LPS addition. In contrast, theaspirated medium containing MBI 29 could depress the ability of freshRAW cells to induce TNF in response to 10 ng or 100 ng of E. coli LPS by98.5% and 75% respectively. Up to 50 μg of MBI 29 caused no apparentdecrease in RAW cell viability as judged by Trypan blue exclusion.

EXAMPLE 6 PROTECTION FROM LETHAL LPS ENDOTOXICITY IN A MOUSE ENDOTOXICSHOCK MODEL

The ability of MBI 29 and MBI 26 to protect against LPS-inducedendotoxemia is assessed in vivo. Mice (8-10 weeks old) are injectedintraperitoneally with 20 μg D-galactosamine (Dgal) to sensitize them toLPS according to the method of Galanos (Galanos, et al., Proc. Natl.Acad Sci., USA, 76:5939-5943, 1979), followed by 0, 50, 100, or 200 μgMBI 29 or MBI 26 in 100 μl. Immediately afterwards LPS (10 or 20 μg) in100 μl is injected. The mice are observed at 24 and 48 hours afterinjections and survivors noted. MBI 26 was shown to be non-toxic up to30.6 mg/kg in mice.

To demonstrate that survival is associated with a reduction in TNFlevels, 10 μg of LPS and 20 mg of Dgal are injected at time 0. Thirtyminutes later, the mice are sacrificed and the blood is taken andcentrifuged to separate the serum which was used in the cell cytotoxicL929 assay. The results suggest that the bactolysins have potential intherapy against endotoxin-associated disorders.

EXAMPLE 7 CHARACTERIZATION OF DERIVATIVE PEPTIDES

Peptides 490-493 (SEQ ID NO:5-8) were found to possess comparable antimicrobial activity to MBI 26 and MBI 29 (Table 6). These peptides do notlyse Red Blood Cells at concentration below 55 μg/mL.

                  TABLE 6                                                         ______________________________________                                        MIC (μg/mL)   RBC Lysis                                                    Peptide                                                                             E. coli (UB 1005)                                                                            P. aeruginosa (H187)                                                                        (μg/mL)                                 ______________________________________                                        MBI 26                                                                              1.1            4.5           >55                                        MBI 29                                                                                0.5                 1.0        >55                                    490      1                  8           >55                                   491      2                  8           >55                                   492      2.4                4.8        >55                                    493      5                  10         >55                                    ______________________________________                                    

α Helical Content of Cationic Peptides by CD Spectra

The α helical content of the peptides were measured in buffer, liposomesand 20% Hexafluoroisopropanol (HFPIP). The liposomes constituted of POPCalone or POPC and POPC and 30% POPG (Table 7)

                  TABLE 7                                                         ______________________________________                                        % α helicity                                                                             POPC       POPC/POPG                                         Peptide  Buffer  Liposomes  Liposomes                                                                             20% HFIP                                  ______________________________________                                        MBI 26   0        0          8      70                                        MBI 29   0       16          8      50                                        490      0       29         27      41                                        491      0        0         15      44                                        492      0       42         19      48                                        493      0       18         20      42                                        ______________________________________                                    

EXAMPLE 8 SYNERGISM BETWEEN CATIONIC PEPTIDES AND ANTIBIOTICS

Synergy between the peptides of the invention and conventionalantibiotics from several different classes was determined by MIC and FICvalues. The following Tables 8-47 show the MIC and FIC values for MBI 26and MBI 29 with different bacteria (A. calcoaceticus, P. aeruginosa, E.cloacae and X. maltophila) and different classes of antibiotics.Synergism is identified as an FIC of about ≦0.5.

                  TABLE 8                                                         ______________________________________                                        Synergy of MBI-26 + Ciprofloxacin against A. calcoaceticus                    Isolate          MIC (μg · mL.sup.-1)                                                             Lowest FIC MBI-26                             Number                                                                              Strain Number                                                                            MBI-26  Ciprofloxacin                                                                          and Ciprofloxacin                           ______________________________________                                        1     ATCC 23055 128     0.5      0.51                                        2     23         32      0.5      0.75                                        3     27         64      0.5      0.75                                        4     28         16      8        0.53                                        5     8220       64      2        0.5                                         6     8221       64      1        0.5                                         7     8222       64      1        0.5                                         8     8223       64      2        0.38                                        9     8224       64      2        0.5                                         10    8225       64      2        0.38                                        11    8226       64      2        0.38                                        ______________________________________                                    

                  TABLE 9                                                         ______________________________________                                        Synergy of MBI-29 + Ciprofloxacin against A. calcoaceticus                    Isolate          MIC (μg · mL.sup.-1)                                                             Lowest FIC MBI-29                             Number                                                                              Strain Number                                                                            MBI-29  Ciprofloxacin                                                                          and Ciprofloxacin                           ______________________________________                                        1     ATCC 23055 32      0.5      0.51                                        2     23         32      0.5      0.75                                        3     27         32      8        0.63                                        4     28         32      8        0.63                                        5     8220       16      2        0.56                                        6     8221       16      0.5      1                                           7     8222       16      2        0.75                                        8     8223       16      2        0.75                                        9     8224       16      1        1                                           10    8225       16      2        0.75                                        11    8226       16      2        0.75                                        ______________________________________                                    

                  TABLE 10                                                        ______________________________________                                        Synergy of MBI-26 + Imipenem against A. calcoaceticus                         Isolate          MIC (μg · mL.sup.-1)                                                             Lowest FIC MBI-26                             Number                                                                              Strain Number                                                                            MBI-26  Imipenem and Imipenem                                ______________________________________                                        1     ATCC 23055 128     2        1.0                                         2     23         64      512      0.13                                        3     27         32      512      0.14                                        4     28         32      16       0.25                                        5     8220       32      2        0.53                                        6     8221       64      1        1.01                                        7     8222       64      4        0.31                                        8     8223       32      2        0.75                                        9     8224       64      2        0.63                                        10    8225       64      2        0.63                                        11    8226       64      4        0.5                                         ______________________________________                                    

                  TABLE 11                                                        ______________________________________                                        Synergy of MBI-29 + Imipenem against A. calcoaceticus                         Isolate          MIC (μg · mL.sup.-1)                                                             Lowest FIC MBI-29                             Number                                                                              Strain Number                                                                            MBI-29  Imipenem and Imipenem                                ______________________________________                                        1     ATCC 23055 64      2        1                                           2     23         32      512      0.13                                        3     27          8      512      0.5                                         4     28         32      8        0.5                                         5     8220        8      2        1                                           6     8221        8      1        1.03                                        7     8222       32      2        0.75                                        8     8223       16      2        1                                           9     8224       32      2        0.51                                        10    8225       32      2        0.51                                        11    8226       32      2        1                                           ______________________________________                                    

                  TABLE 12                                                        ______________________________________                                        Synergy of MBI-29 + Tobramycin against A. calcoaceticus                       Isolate          MIC (μg · mL.sup.-1)                                                             Lowest FIC MBI-26                             Number                                                                              Strain Number                                                                            MBI-26  Tobramycin                                                                             and Tobramycin                              ______________________________________                                        1     ATCC 23055 64      4        0.31                                        2     23         32      16       ND                                          3     27         32      1        1.01                                        4     28         32      128      0.26                                        5     8220       64      4        0.5                                         6     8221       64      4        0.5                                         7     8222       64      4        0.5                                         8     8223       64      4        0.5                                         9     8224       64      4        0.5                                         10    8225       64      4        0.5                                         11    8226       64      4        0.5                                         ______________________________________                                    

                  TABLE 13                                                        ______________________________________                                        Synergy of MBI-29 + Tobramycin against A. calcoaceticus                       Isolate          MIC (μg · mL.sup.-1)                                                             Lowest FIC MBI-29                             Number                                                                              Strain Number                                                                            MBI-29  Tobramycin                                                                             and Tobramycin                              ______________________________________                                        1     ATCC 23055 32      2        0.75                                        2     23          4      4        1.25                                        3     27          8      1        1.03                                        4     28         32      128      0.26                                        5     8220       32      4        0.5                                         6     8221       32      4        0.5                                         7     8222       32      4        0.5                                         8     8223       32      4        0.5                                         9     8224       32      4        0.5                                         10    8225       32      4        0.5                                         11    8226       32      4        0.5                                         ______________________________________                                    

                  TABLE 14                                                        ______________________________________                                        Synergy of MBI-29 + Piperacillin against A. calcoaceticus                     Isolate          MIC (μg · mL.sup.-1)                                                             Lowest FIC MBI-26                             Number                                                                              Strain Number                                                                            MBI-26  Piperacillin                                                                           and Piperacillin                            ______________________________________                                        1     ATCC 23055 64       8       0.75                                        2     23         32      1024     0.26                                        3     27         32      16       0.38                                        4     28         16       4       1.03                                        5     8220       64      16       0.38                                        6     8221       64      16       0.38                                        7     8222       64      32       0.25                                        8     8223       64      128      0.13                                        9     8224       64      256      0.16                                        10    8225       64      32       0.25                                        11    8226       64      32       0.25                                        ______________________________________                                    

                  TABLE 15                                                        ______________________________________                                        Synergy of MBI-29 + Piperacillin against A. calcoaceticus                     Isolate          MIC (μg · mL.sup.-1)                                                             Lowest FIC MBI-29                             Number                                                                              Strain Number                                                                            MBI-29  Piperacillin                                                                           and Piperacillin                            ______________________________________                                        1     ATCC 23055 32       8       1                                           2     23         16      1024     0.28                                        3     27         16      16       0.5                                         4     28          8       4       1.03                                        5     8220       32      16       0.5                                         6     8221       32      16       0.38                                        7     8222       32      32       0.25                                        8     8223       32      32       0.25                                        9     8224       32      128      0.19                                        10    8225       32      32       0.25                                        11    8226       32      32       0.25                                        ______________________________________                                    

                  TABLE 16                                                        ______________________________________                                        Synergy of MBI-26 + Ceftazidime against A. calcoaceticus                      Isolate          MIC (μg · mL.sup.-1)                                                             Lowest FIC MBI-26                             Number                                                                              Strain Number                                                                            MBI-26  Ceftazidime                                                                            and Ceftazidime                             ______________________________________                                        1     ATCC 23055 128      2       1.01                                        2     23         64      128      0.25                                        3     27         32      32       0.38                                        4     28         32      16       0.38                                        5     8220       32      16       0.38                                        6     8221       64       8       0.25                                        7     8222       64      16       0.5                                         8     8223       32      16       0.25                                        9     8224       64      64       0.25                                        10    8225       64      32       0.31                                        11    8226       64      64       0.38                                        ______________________________________                                    

                  TABLE 17                                                        ______________________________________                                        Synergy of MBI-29 + Ceftazidime against A. calcoaceticus                      Isolate          MIC (μg · mL.sup.-1)                                                             Lowest FIC MBI-29                             Number                                                                              Strain Number                                                                            MBI-29  Ceftazidime                                                                            and Ceftazidime                             ______________________________________                                        1     ATCC 23055 64       4       0.63                                        2     23         32      128      0.26                                        3     27          8       4       1.5                                         4     28         32      16       0.63                                        5     8220        8       8       1.03                                        6     8221       16       8       1                                           7     8222       32      16       0.38                                        8     8223       16      16       0.75                                        9     8224       32      64       0.25                                        10    8225       32       8       0.5                                         11    8226       32      16       0.38                                        ______________________________________                                    

                  TABLE 18                                                        ______________________________________                                        Synergy of MBI-29 + Ciprofloxacin against E. cloacae                          Isolate          MIC (μg · mL.sup.-1)                                                             Lowest FIC MBI-26                             Number                                                                              Strain Number                                                                            MBI-26  Ciprofloxacin                                                                          and Ciprofloxacin                           ______________________________________                                        12    ATCC 13047 64      0.25     1.01                                        13    37-1       256     1        0.26                                        14    938-2      64      1        0.38                                        15    B5546      64      1        0.38                                        16    R4148      64      2        0.38                                        17    R4148-2    64      2        0.38                                        18    3077       64      0.5      0.75                                        19    3356       128     0.25     1                                           20    14269      256     1        0.28                                        21    14661      512     1        0.28                                        22    18801      32      1        0.38                                        ______________________________________                                    

                  TABLE 19                                                        ______________________________________                                        Synergy of MBI-29 + Ciprofloxacin against E. cloacae                          Isolate          MIC (μg · mL.sup.-1)                                                             Lowest FIC MBI-29                             Number                                                                              Strain Number                                                                            MBI-29  Ciprofloxacin                                                                          and Ciprofloxacin                           ______________________________________                                        12    ATCC 13047 32      0.25     1.01                                        13    37-1       32      1        1                                           14    938-2      32      1        0.5                                         15    B5546      32      1        0.5                                         16    R4148      32      2        0.63                                        17    R4148-2    16      2        0.75                                        18    3077       32      1        0.75                                        19    3356       32      0.25     1.01                                        20    14269      32      1        0.75                                        21    14661      32      1        0.75                                        22    18801      32      1        0.75                                        ______________________________________                                    

                  TABLE 20                                                        ______________________________________                                        Synergy of MBI-26 + Imipenem against E. cloacae                               Isolate          MIC (μg · mL.sup.-1)                                                           Lowest FIC                                      Number                                                                              Strain Number                                                                            MBI-26  Imipenem                                                                             MBI-26 and Imipenem                           ______________________________________                                        12    ATCC 13047 16      1      1                                             13    37-1       64      2      0.75                                          14    938-2      64      4      0.75                                          15    B5546      64      2      1                                             16    R4148      64      4      0.75                                          17    R4148-2    32      2      1                                             18    3077       64      4      0.75                                          19    3356       64      4      0.75                                          20    14269      64      4      0.51                                          21    14661      64      2      1.01                                          22    18801      64      2      1.01                                          ______________________________________                                    

                  TABLE 21                                                        ______________________________________                                        Synergy of MBI-29 + Imipenem against E. cloacae                               Isolate          MIC (μg · mL.sup.-1)                                                            Lowest FIC                                     Number                                                                              Strain Number                                                                            MBI-269  Imipenem                                                                             MBI-29 and Imipenem                          ______________________________________                                        12    ATCC 13047 64       2      1                                            13    37-1       128      2      1                                            14    938-2      32       2      1.5                                          15    B5546      32       2      0.75                                         16    R4148      64       4      0.56                                         17    R4148-2    32       2      0.75                                         18    3077       32       2      1                                            19    3356       32       2      1.5                                          20    14269      128      2      1                                            21    14661      64       2      1                                            22    18801      64       2      1                                            ______________________________________                                    

                  TABLE 22                                                        ______________________________________                                        Synergy of MBI-26 + Tobramycin against E. cloacae                             Isolate          MIC (μg · mL.sup.-1)                                                             Lowest FIC MBI-26                             Number                                                                              Strain Number                                                                            MBI-26   Tobramycin                                                                            and Tobramycin                              ______________________________________                                        12    ATCC 13047 64       1       1.01                                        13    37-1       64       1       1.01                                        14    938-2      64       2       1.01                                        15    B5546      32       2       0.51                                        16    R4148      32       1       1.06                                        17    R4148-2    64       1       ND                                          18    3077       64       1       1.06                                        19    3356       64       1       1.01                                        20    14269      64       1       1.01                                        21    14661      64       1       1.01                                        22    18801      32       1       1.01                                        ______________________________________                                    

                  TABLE 23                                                        ______________________________________                                        Synergy of MBI-29 + Tobramycin against E. cloacae                             Isolate          MIC (μg · mL.sup.-1)                                                             Lowest FIC MBI-29                             Number                                                                              Strain Number                                                                            MBI-29   Tobramycin                                                                            and Tobramycin                              ______________________________________                                        12    ATCC 13047 32       1       1                                           13    37-1       32       1       1                                           14    938-2      16       1       1.01                                        15    B5546      32       1       1.01                                        16    R4148      16       1       1.03                                        17    R4148-2    16       1       1.01                                        18    3077       32       1       1.01                                        19    3356       32       1       1.01                                        20    14269      16       1       1.01                                        21    14661      32       1       1.01                                        22    18801      16       1       1.01                                        ______________________________________                                    

                  TABLE 24                                                        ______________________________________                                        Synergy of MBI-26 + Piperacillin against E. cloacae                           Isolate          MIC (μg · mL.sup.-1)                                                             Lowest FIC MBI-26                             Number                                                                              Strain Number                                                                            MBI-26   Piperacillin                                                                          and Piperacillin                            ______________________________________                                        12    ATCC 13047 64       8       0.75                                        13    37-1       64       4       1.01                                        14    938-2      64       8       0.51                                        15    B5546      64       8       0.56                                        16    R4148      64       1024    0.13                                        17    R4148-2    64       1024    0.25                                        18    3077       64       8       0.56                                        19    3356       64       64      0.25                                        20    14269      512      1024    0.03                                        21    14661      64       16      0.5                                         22    18801      32       4       1.01                                        ______________________________________                                    

                  TABLE 25                                                        ______________________________________                                        Synergy of MBI-29 + Piperacillin against E. cloacae                           Isolate          MIC (μg · mL.sup.-1)                                                             Lowest FIC MBI-29                             Number                                                                              Strain Number                                                                            MBI-29   Piperacillin                                                                          and Piperacillin                            ______________________________________                                        12    ATCC 13047 32       8       0.75                                        13    37-1       32       4       1.01                                        14    938-2      32       4       1.01                                        15    B5546      32       8       0.63                                        16    R4148      32       1024    0.28                                        17    R4148-2    32       1024    0.28                                        18    3077       32       4       1.01                                        19    3356       32       64      0.31                                        20    14269      128      1024    0.07                                        21    14661      32       8       1.01                                        22    18801      16       4       1.01                                        ______________________________________                                    

                  TABLE 26                                                        ______________________________________                                        Synergy of MBI-26 + Ceftazidime against E. cloacae                            Isolate          MIC (μg · mL.sup.-1)                                                             Lowest FIC MBI-26                             Number                                                                              Strain Number                                                                            MBI-26   Ceftazidime                                                                           and Ceftazidime                             ______________________________________                                        12    ATCC 13047 16       4       0.75                                        13    37-1       256      2       1                                           14    938-2      64       2       1.01                                        15    B5546      64       2       1.01                                        16    R4148      64       512     0.25                                        17    R4148-2    32       512     31                                          18    3077       64       2       1.01                                        19    3356       128      128     0.19                                        20    14269      512      512     0.03                                        21    14661      512      512     0.03                                        22    18801      256      2       1                                           ______________________________________                                    

                  TABLE 27                                                        ______________________________________                                        Synergy of MBI-29 + Ceftazidime against E. cloacae                            Isolate          MIC (μg · mL.sup.-1)                                                             Lowest FIC MBI-29                             Number                                                                              Strain Number                                                                            MBI-29   Ceftazidime                                                                           and Ceftazidime                             ______________________________________                                        12    ATCC 13047 64       4       0.53                                        13    37-1       128      2       1                                           14    938-2      32       2       1.01                                        15    B5546      32       2       1.01                                        16    R4148      64       512     0.16                                        17    R4148-2    32       512     0.28                                        18    3077       32       2       1.01                                        19    3356       32       128     0.3                                         20    14269      128      512     0.09                                        21    14661      128      512     0.09                                        22    18801      128      2       1                                           ______________________________________                                    

                  TABLE 28                                                        ______________________________________                                        Synergy of MBI-26 + Ciprofloxacin against P. aeruginosa                       Isolate          MIC (μg · mL.sup.-1)                                                             Lowest FIC MBI-26                             Number                                                                              Strain Number                                                                            MBI-26  Ciprofloxacin                                                                          and Ciprofloxacin                           ______________________________________                                        23    ATCC 27853 64      1        0.56                                        24    34         64      1        0.53                                        25    923-1      64      0.25     1.01                                        26    3101       64      0.25     1.01                                        27    14644      256     32       ND                                          28    15036      256     16       1                                           29    15545      64      0.25     1.01                                        30    B3999-1    64      0.25     1.01                                        31    U7688-1    64      0.25     1.01                                        32    W2897-1    64      0.25     1.01                                        33    W5483-2    64      0.25     1.01                                        ______________________________________                                    

                  TABLE 29                                                        ______________________________________                                        Synergy of MBI-29 + Ciprofloxacin against P. aeruginosa                       Isolate          MIC (μg · mL.sup.-1)                                                             Lowest FIC MBI-29                             Number                                                                              Strain Number                                                                            MBI-29  Ciprofloxacin                                                                          and Ciprofloxacin                           ______________________________________                                        23    ATCC 27853 32      1        0.75                                        24    34         32      1        0.75                                        25    923-1      32      0.25     1.01                                        26    3101       32      0.25     1.01                                        27    14644      128     32       ND                                          28    15036      128     32       ND                                          29    15545      32      0.25     0.63                                        30    B3999-1    32      0.25     1.01                                        31    U7688-1    32      0.25     1.01                                        32    W2897-1    32      0.25     1.01                                        33    W5483-2    32      0.25     1.01                                        ______________________________________                                    

                  TABLE 30                                                        ______________________________________                                        Synergy of MBI-26 + Imipenem against P. aeruginosa                            Isolate          MIC (μg · mL.sup.-1)                                                             Lowest FIC MBI-26                             Number                                                                              Strain Number                                                                            MBI-26  Imipenem and Imipenem                                ______________________________________                                        23    ATCC 27853 64      8        0.5                                         24    34         128     512      0.38                                        25    923-1      64      4        1.01                                        26    3101       64      8        0.75                                        27    14644      64      16       0.51                                        28    15036      64      16       1                                           29    15545      64      128      ND                                          30    B3999-1    64      128      ND                                          31    U7688-1    64      16       1                                           32    W2897-1    64      128      0.25                                        33    W5483-2    64      64       ND                                          ______________________________________                                    

                  TABLE 31                                                        ______________________________________                                        Synergy of MBI-29 + Imipenem against P. aeruginosa                            Isolate          MIC (μg · mL.sup.-1)                                                             Lowest FIC MBI-29                             Number                                                                              Strain Number                                                                            MBI-29  Imipenem and Imipenem                                ______________________________________                                        23    ATCC 27853 64       4       0.75                                        24    34         32      512      0.16                                        25    923-1      64       2       1.13                                        26    3101       32       4       0.75                                        27    14644      64       8       1                                           28    15036      64       8       1                                           29    15545      32      256      0.63                                        30    B3999-1    64      256      1                                           31    U7688-1    64      256      0.75                                        32    W2897-1    32      256      0.75                                        33    W5483-2    64      256      0.5                                         ______________________________________                                    

                  TABLE 32                                                        ______________________________________                                        Synergy of MBI-26 + Tobramycin against P. aeruginosa                          Isolate          MIC (μg · mL.sup.-1)                                                             Lowest FIC MBI-26                             Number                                                                              Strain Number                                                                            MBI-26  Tobramycin                                                                             and Tobramycin                              ______________________________________                                        23    ATCC 27853 64      1        1.01                                        24    34         128     128      0.5                                         25    923-1      64      1        1.01                                        26    3101       64      1        1.01                                        27    14644      64      2        0.53                                        28    15036      64      1        1.01                                        29    15545      64      2        1                                           30    B3999-1    64      2        0.51                                        31    U7688-1    64      1        1.01                                        32    W2897-1    64      1        1.01                                        33    W5483-2    64      1        1.01                                        ______________________________________                                    

                  TABLE 33                                                        ______________________________________                                        Synergy of MBI-29 + Tobramycin against P. aeruginosa                          Isolate          MIC (μg · mL.sup.-1)                                                             Lowest FIC MBI-29                             Number                                                                              Strain Number                                                                            MBI-29  Tobramycin                                                                             and Tobramycin                              ______________________________________                                        23    ATCC 27853 32      1        1.01                                        24    34         16      64       0.5                                         25    923-1      32      1        1.01                                        26    3101       32      1        1.01                                        27    14644      32      1        1.01                                        28    15036      32      1        1.01                                        29    15545      32      2        0.75                                        30    B3999-1    32      1        1.01                                        31    U7688-1    32      2        0.56                                        32    W2897-1    32      1        1.01                                        33    W5483-2    32      2        0.51                                        ______________________________________                                    

                  TABLE 34                                                        ______________________________________                                        Synergy of MBI-26 + Piperacillin against P. aeruginosa                        Isolate          MIC (μg · mL.sup.-1)                                                             Lowest FIC MBI-26                             Number                                                                              Strain Number                                                                            MBI-26  Piperacillin                                                                           and Piperacillin                            ______________________________________                                        23    ATCC 27853 128     256      0.19                                        24    34         128     1024     0.5                                         25    923-1       64      64      0.5                                         26    3101        64      64      0.63                                        27    14644       64     512      ND                                          28    15036      256     1024     0.09                                        29    15545      256     512      0.14                                        30    B3999-1    256     512      0.25                                        31    U7688-1    256     512      ND                                          32    W2897-1     64      64      1                                           33    W5483-2    256     128      1                                           ______________________________________                                    

                  TABLE 35                                                        ______________________________________                                        Synergy of MBI-29 + Piperacillin against P. aeruginosa                        Isolate          MIC (μg · mL.sup.-1)                                                             Lowest FIC MBI-29                             Number                                                                              Strain Number                                                                            MBI-29  Piperacillin                                                                           and Piperacillin                            ______________________________________                                        23    ATCC 27853 64      128      ND                                          24    34         32      1024     0.28                                        25    923-1      32       32      0.5                                         26    3101       32      128      0.31                                        27    14644      128     1024     ND                                          28    15036      128     1024     0.09                                        29    15545      64      512      0.16                                        30    B3999-1    128     256      0.12                                        31    U7688-1    32      256      0.5                                         32    W2897-1    32       32      0.75                                        33    W5483-2    128     512      0.19                                        ______________________________________                                    

                  TABLE 36                                                        ______________________________________                                        Synergy of MBI-26 + Ceftazidime against P. aeruginosa                         Isolate          MIC (μg · mL.sup.-1)                                                             Lowest FIC MBI-26                             Number                                                                              Strain Number                                                                            MBI-26  Ceftazidime                                                                            and Ceftazidime                             ______________________________________                                        23    ATCC 27853 128     32       0.31                                        24    34         128     32       0.75                                        25    923-1      128      4       1.01                                        26    3101       128      8       0.56                                        27    14644      256     128      1                                           28    15036      256      2       2                                           29    15545      256      8       0.53                                        30    B3999-1    256     16       1.01                                        31    U7688-1    256     32       0.25                                        32    W2897-1    256     32       0.26                                        33    W5483-2    256      8       1.01                                        ______________________________________                                    

                  TABLE 37                                                        ______________________________________                                        Synergy of MBI-29 + Ceftazidime against P. aeruginosa                         Isolate          MIC (μg · mL.sup.-1)                                                            Lowest FIC MBI-29                              Number                                                                              Strain Number                                                                            MBI-29  Ceftazidime                                                                           and Ceftazidime                              ______________________________________                                        23    ATCC 27853 64      32      0.38                                         24    34         32      64      0.31                                         25    923-1      64      4       0.56                                         26    3101       64      8       0.56                                         27    14644      128     256     1                                            28    15036      128     4       0.51                                         29    15545      64      8       0.5                                          30    B3999-1    128     8       0.31                                         31    U7688-1    128     8       0.63                                         32    W2897-1    64      4       0.51                                         33    W5483-2    128     8       0.56                                         ______________________________________                                    

                  TABLE 38                                                        ______________________________________                                        Synergy of MBI-26 + Ciprofloxacin against X. maltophila                                                       Lowest FIC                                    Isolate          MIC (μg · mL.sup.-1)                                                             MBI-26 and                                    Number                                                                              Strain Number                                                                            MBI-26  Ciprofloxacin                                                                          Ciprofloxacin                               ______________________________________                                        34    ATCC 13637 64      0.5     0.63                                         35    6          64      4       0.38                                         36    16         64      8       1                                            37    3095       64      16      1                                            38    7901       256     32      ND                                           39    B3110.1    64      2       2.01                                         40    R1302-1    128     4       1                                            41    R4230      64      2       1                                            42    TF29-1     128     8       ND                                           43    TF29-2     128     8       1                                            44    W6896-2    256     4       0.13                                         45    W11291     64      2       2.01                                         ______________________________________                                    

                  TABLE 39                                                        ______________________________________                                        Synergy of MBI-29 + Ciprofloxacin against X. maltophila                                                       Lowest FIC                                    Isolate          MIC (μg · mL.sup.-1)                                                             MBI-29 and                                    Number                                                                              Strain Number                                                                            MBI-29  Ciprofloxacin                                                                          Ciprofloxacin                               ______________________________________                                        34    ATCC 13637 32      0.5     1.01                                         35    6          32      2       0.38                                         36    16         32      8       ND                                           37    3095       64      16      0.5                                          38    7901       64      32      0.75                                         39    B3110.1    32      2       1.01                                         40    R1302-1    32      4       0.56                                         41    R4230      16      4       0.5                                          42    TF29-1     16      8       1                                            43    TF29-2     16      8       0.53                                         44    W6896-2    16      8       1                                            45    W11291     32      4       0.75                                         ______________________________________                                    

                  TABLE 40                                                        ______________________________________                                        Synergy of MBI-26 + Imipenem against X. maltophila                            Isolate          MIC (μg · mL.sup.-1)                                                            Lowest FIC MBI-26                              Number                                                                              Strain Number                                                                            MBI-26  Imipenem                                                                              and Imipenem                                 ______________________________________                                        34    ATCC 13637  64     512     0.13                                         35    6          128     512     0.16                                         36    16         256     512     ND                                           37    3095       512     512     ND                                           38    7901       256     512     ND                                           39    B3110.1    256     512     ND                                           40    R1302-1    128     512     ND                                           41    R4230       64     512     ND                                           42    TF29-1     128     512     ND                                           43    TF29-2     128     512     ND                                           44    W6896-2    256     512     0.25                                         45    W11291     512     512     ND                                           ______________________________________                                    

                  TABLE 41                                                        ______________________________________                                        Synergy of MBI-29 + Imipenem against X. maltophila                            Isolate          MIC (μg · mL.sup.-1)                                                            Lowest FIC MBI-29                              Number                                                                              Strain Number                                                                            MBI-29  Imipenem                                                                              and Imipenem                                 ______________________________________                                        34    ATCC 13637 32      512     0.19                                         35    6          32      512     0.25                                         36    16         32      512     0.3                                          37    3095       64      512     ND                                           38    7901       64      512     0.38                                         39    R3110.1    32      512     ND                                           40    R1302-1    64      512     0.25                                         41    R4230      32      512     0.5                                          42    TF29-1     16      512     0.56                                         43    TF29-2     16      512     0.56                                         44    W6896-2    64      512     0.38                                         45    W11291     64      512     0.38                                         ______________________________________                                    

                  TABLE 42                                                        ______________________________________                                        Synergy of MBI-26 + Tobramycin against X. maltophila                          Isolate          MIC (μg · mL.sup.-1)                                                            Lowest FIC MBI-26                              Number                                                                              Strain Number                                                                            MBI-26  Tobramycin                                                                            and Tobramycin                               ______________________________________                                        34    ATCC 13637 64      32      0.16                                         35    6          64       4      0.25                                         36    16         256     128     0.63                                         37    3095       32      32      0.5                                          38    7901       32      32      1.01                                         39    B3110.1    256     32      0.63                                         40    R1302-1    128     32      0.63                                         41    R4230      64      16      0.38                                         42    TF29-1     32       2      1.01                                         43    TF29-2     32       2      1.01                                         44    W6896-2    32      16      0.5                                          45    W11291     512     128     ND                                           ______________________________________                                    

                  TABLE 43                                                        ______________________________________                                        Synergy of MBI-29 + Tobramycin against X. maltophila                          Isolate          MIC (μg · mL.sup.-1)                                                            Lowest FIC MBI-29                              Number                                                                              Strain Number                                                                            MBI-29  Tobramycin                                                                            and Tobramycin                               ______________________________________                                        34    ATCC 13637  8       8      0.63                                         35    6          16       4      0.75                                         36    16         32      128     0.31                                         37    3095       64      32      0.16                                         38    7901       64      32      0.28                                         39    B3110.1    32      16      0.56                                         40    R1302-1    64      32      0.53                                         41    R4230      16      16      0.56                                         42    TF29-1      8       1      1.03                                         43    TF29-2     16       1      1.5                                          44    W6896-2    16       8      0.63                                         45    W11291     64      128     ND                                           ______________________________________                                    

                  TABLE 44                                                        ______________________________________                                        Synergy of MBI-26 + Piperacillin against X. maltophila                        Isolate          MIC (μg · mL.sup.-1)                                                            Lowest FIC MBI-26                              Number                                                                              Strain Number                                                                            MBI-26  Piperacillin                                                                          and Piperacillin                             ______________________________________                                        34    ATCC 13637 64      1024    0.14                                         35    6          64       512    ND                                           36    16         256     1024    ND                                           37    3095       64       512    ND                                           38    7901       64       512    ND                                           39    B3110.1    64       512    ND                                           40    R1302-1    64       512    ND                                           41    R4230      64      1024    0.5                                          42    TF29-1     128     1024    0.2                                          43    TF29-2     128     1024    0.5                                          44    W6896-2    32      1024    0.6                                          45    W11291     64       512    ND                                           ______________________________________                                    

                  TABLE 45                                                        ______________________________________                                        Synergy of MBI-29 + Piperacillin against X. maltophila                        Isolate          MIC (μg · mL.sup.-1)                                                            Lowest FIC MBI-29                              Number                                                                              Strain Number                                                                            MBI-29  Piperacillin                                                                          and Piperacillin                             ______________________________________                                        34    ATCC 13637 32      1024    0.19                                         35    6          32       64     0.19                                         36    16         32      1024    0.38                                         37    3095       64      1024    0.38                                         38    7901       64      1024    0.28                                         39    B3110.1    32      1024    0.63                                         40    R1302-1    64      1024    0.25                                         41    R4230      32      1024    0.3                                          42    TF29-1     32      1024    0.2                                          43    TF29-2     32      1024    0.28                                         44    W6896-2    64      1024    0.13                                         45    W11291     64      1024    0.31                                         ______________________________________                                    

                  TABLE 46                                                        ______________________________________                                        Synergy of MBI-26 + Ceftazidime against X. maltophila                         Isolate          MIC (μg · mL.sup.-1)                                                            Lowest FIC MBI-26                              Number                                                                              Strain Number                                                                            MBI-26  Ceftazidime                                                                           and Ceftazidime                              ______________________________________                                        34    ATCC 13637  64     64      0.28                                         35    6          128     64      0.25                                         36    16         256     32      0.51                                         37    3095       512     512     ND                                           38    7901       256     128     1                                            39    B3110.1    256     64      1.13                                         40    R1302-1    128     16      1                                            41    R4230       64      4      1                                            42    TF29-1     128      4      1                                            43    TF29-2     128      4      1.01                                         44    W6896-2    256     64      1                                            45    W11291     512     128     1                                            ______________________________________                                    

                  TABLE 47                                                        ______________________________________                                        Synergy of MBI-29 + Ceftazidime against X. maltophila                         Isolate          MIC (μg · mL.sup.-1)                                                            Lowest FIC MBI-29                              Number                                                                              Strain Number                                                                            MBI-29  Ceftazidime                                                                           and Ceftazidime                              ______________________________________                                        34    ATCC 13637 32      64      0.28                                         35    6          32      32      0.31                                         36    16         32      16      0.63                                         37    3095       64      512     ND                                           38    7901       64      256     0.31                                         39    B3110.1    32      128     0.75                                         40    R1302-1    64      32      0.25                                         41    R4230      32       8      0.5                                          42    TF29-1     16       4      1                                            43    TF29-2     16       2      1.01                                         44    W6896-2    64      64      0.5                                          45    W11291     64      256     0.28                                         ______________________________________                                    

Note: MBI 26 (1.25 μg/mL) also showed synergy with Lysozyme (>64×reduction in MIC), Novobiocin (>4× reduction in MIC), Nalidixic acid (2×reduction in MIC) and carbenicillin (2× reduction in MIC).

Although the invention has been described with reference to thepresently preferred embodiment, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 37                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 26 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - Lys Trp Lys Ser Phe Ile Lys Lys Leu Thr Th - #r Ala Val Lys Lys Val         #                 15                                                          - Leu Thr Thr Gly Leu Pro Ala Leu Ile Ser                                     #             25                                                              - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 26 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Lys Trp Lys Ser Phe Ile Lys Lys Leu Thr Se - #r Ala Ala Lys Lys Val         #                 15                                                          - Val Thr Thr Ala Lys Pro Leu Ile Ser Ser                                     #             25                                                              - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 26 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - Lys Trp Lys Ser Phe Ile Lys Asn Leu Thr Ly - #s Gly Gly Ser Lys Ile         #                 15                                                          - Leu Thr Thr Gly Leu Pro Ala Leu Ile Ser                                     #             25                                                              - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 26 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Lys Trp Lys Lys Phe Ile Lys Asn Leu Thr Ly - #s Gly Gly Ser Lys Ile         #                 15                                                          - Leu Thr Thr Gly Leu Pro Ala Leu Ile Ser                                     #             25                                                              - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 26 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 - Lys Trp Lys Ser Phe Ile Lys Asn Leu Glu Ly - #s Val Leu Lys Pro Gly         #                 15                                                          - Gly Leu Leu Ser Asn Ile Val Thr Ser Leu                                     #             25                                                              - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 26 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 - Lys Trp Lys Ser Phe Ile Lys Asn Leu Glu Ly - #s Val Leu Lys Lys Gly         #                 15                                                          - Pro Ile Leu Ala Asn Leu Val Ser Ile Val                                     #             25                                                              - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 26 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - Lys Trp Lys Glu Phe Ile Lys Lys Leu Thr Th - #r Ala Val Lys Lys Val         #                 15                                                          - Leu Thr Thr Gly Leu Pro Ala Leu Ile Ser                                     #             25                                                              - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 26 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 - Lys Trp Lys Lys Phe Ile Lys Glu Leu Gln Ly - #s Val Leu Ala Pro Gly         #                 15                                                          - Gly Leu Leu Ser Asn Ile Val Thr Ser Leu                                     #             25                                                              - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 26 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 - Lys Trp Lys Ser Phe Ile Lys Lys Leu Thr Se - #r Val Leu Lys Lys Val         #                 15                                                          - Val Thr Thr Ala Leu Pro Ala Leu Ile Ser                                     #             25                                                              - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 26 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                - Lys Trp Lys Ser Phe Ile Lys Asn Leu Thr Ly - #s Val Leu Lys Lys Val         #                 15                                                          - Val Thr Thr Ala Leu Pro Ala Leu Ile Ser                                     #             25                                                              - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 26 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                - Lys Trp Lys Leu Phe Lys Lys Lys Gly Thr Gl - #y Ala Val Leu Thr Val         #                 15                                                          - Leu Thr Thr Gly Leu Pro Ala Leu Ile Ser                                     #             25                                                              - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 26 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                - Lys Trp Lys Ser Phe Ile Lys Lys Leu Thr Se - #r Val Leu Lys Lys Val         #                 15                                                          - Val Thr Thr Ala Lys Pro Leu Ile Ser Ser                                     #             25                                                              - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 26 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                - Lys Lys Lys Ser Phe Ile Lys Leu Leu Thr Se - #r Ala Lys Val Ser Val         #                 15                                                          - Leu Thr Thr Ala Lys Pro Leu Ile Ser Ser                                     #             25                                                              - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 26 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                - Lys Trp Lys Lys Phe Ile Lys Glu Leu Gln Ly - #s Val Leu Lys Pro Gly         #                 15                                                          - Gly Leu Leu Ser Asn Ile Val Thr Ser Leu                                     #             25                                                              - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                - Lys Lys Trp Trp Arg Arg Val Leu Ser Gly Le - #u Lys Thr Gly Pro Ala         #                 15                                                          - Leu Ser Asn Val                                                                          20                                                               - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                - Lys Lys Trp Trp Arg Arg Val Leu Lys Gly Le - #u Ser Ser Gly Pro Ala         #                 15                                                          - Leu Ser Asn Val                                                                          20                                                               - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                - Lys Lys Trp Trp Arg Arg Ala Leu Gln Ala Le - #u Lys Asn Gly Pro Ala         #                 15                                                          - Leu Ser Asn Val                                                                          20                                                               - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 24 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                - Lys Lys Trp Trp Arg Arg Val Leu Ser Gly Le - #u Lys Thr Ala Gly Pro         #                 15                                                          - Ala Ile Gln Ser Val Leu Asn Lys                                                          20                                                               - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 24 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                - Lys Lys Trp Trp Arg Arg Ala Leu Gln Gly Le - #u Lys Thr Ala Gly Pro         #                 15                                                          - Ala Ile Gln Ser Val Leu Asn Lys                                                          20                                                               - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 22 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                - Lys Lys Trp Trp Lys Ala Gln Lys Ala Val As - #n Ser Gly Pro Asn Ala         #                 15                                                          - Leu Gln Thr Leu Ala Gln                                                                  20                                                               - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 22 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                - Lys Lys Trp Trp Lys Ala Lys Lys Phe Ala As - #n Ser Gly Pro Asn Ala         #                 15                                                          - Leu Gln Thr Leu Ala Gln                                                                  20                                                               - (2) INFORMATION FOR SEQ ID NO:22:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 23 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                - Lys Lys Trp Trp Lys Phe Ile Lys Lys Ala Va - #l Asn Ser Gly Thr Thr         #                 15                                                          - Gly Leu Gln Thr Leu Ala Ser                                                              20                                                               - (2) INFORMATION FOR SEQ ID NO:23:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                - Lys Lys Ser Phe Phe Lys Lys Leu Thr Ser Va - #l Ala Ser Ser Val Leu         #                 15                                                          - Ser                                                                         - (2) INFORMATION FOR SEQ ID NO:24:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                - Trp Lys Val Phe Lys Ser Phe Ile Lys Lys Al - #a Ser Ser Phe Ala Gln         #                 15                                                          - Ser Val Leu Asp                                                                          20                                                               - (2) INFORMATION FOR SEQ ID NO:25:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 18 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                - Lys Lys Trp Arg Lys Ser Phe Phe Lys Gln Va - #l Gly Ser Phe Asp Asn         #                 15                                                          - Ser Val                                                                     - (2) INFORMATION FOR SEQ ID NO:26:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 26 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                           #8, 10, 11, 14 and 15N: 4, 7,                                                 #where Xaa at positionsORMATION:                                              #8, 10, 11, 14 and 15 are selected from argin - #ine,                                        lysine, g - #lutamic acid, aspartic acid, glutamine,           #serine, histidine and threonine                                              #9, 12 and 13 LOCATION: 5, 6,                                                 #where Xaa at positionsORMATION:                                              #9, 12 and 13 are selected from isoleucine,                                                  valine, l - #eucine, alanine, cysteine, glycine,                              phenylalanin - #e, proline, tryptophan, tyrosine,              #and methioninenorleucine                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                - Lys Trp Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Val         #                 15                                                          - Leu Thr Thr Gly Leu Pro Ala Leu Ile Ser                                     #             25                                                              - (2) INFORMATION FOR SEQ ID NO:27:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 26 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                           #8, 10, 11, 14 and 15N: 4, 7,                                                 #where Xaa at positionsORMATION:                                              #8, 10, 11, 14 and 15 are selected from argin - #ine,                                        lysine, g - #lutamic acid, aspartic acid, glutamine,           #serine, histidine and threonine                                              #9, 12 and 13 LOCATION: 5, 6,                                                 #where Xaa at positionsORMATION:                                              #9, 12 and 13 are selected from isoleucine,                                                  valine, l - #eucine, alanine, cysteine, glycine,                              phenylalanin - #e, proline, tryptophan, tyrosine,              #and methioninenorleucine                                                     -    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                 - Lys Trp Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Val         #                 15                                                          - Val Thr Thr Ala Lys Pro Leu Ile Ser Ser                                     #             25                                                              - (2) INFORMATION FOR SEQ ID NO:28:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 26 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                           #8, 10, 11, 14 and 15N: 4, 7,                                                 #where Xaa at positionsORMATION:                                              #8, 10, 11, 14 and 15 are selected from argin - #ine,                                        lysine, g - #lutamic acid, aspartic acid, glutamine,           #serine, histidine and threonine                                              #9, 12 and 13 LOCATION: 5, 6,                                                 #where Xaa at positionsORMATION:                                              #9, 12 and 13 are selected from isoleucine,                                                  valine, l - #eucine, alanine, cysteine, glycine,                              phenylalanin - #e, proline, tryptophan, tyrosine,              #and methioninenorleucine                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                - Lys Trp Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Ile         #                 15                                                          - Leu Thr Thr Gly Leu Pro Ala Leu Ile Ser                                     #             25                                                              - (2) INFORMATION FOR SEQ ID NO:29:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 26 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                           #8, 10, 11, 14 and 15N: 4, 7,                                                 #where Xaa at positionsORMATION:                                              #8, 10, 11, 14 and 15 are selected from argin - #ine,                                        lysine, g - #lutamic acid, aspartic acid, glutamine,           #serine, histidine and threonine                                              #9, 12 and 13 LOCATION: 5, 6,                                                 #where Xaa at positionsORMATION:                                              #9, 12 and 13 are selected from isoleucine,                                                  valine, l - #eucine, alanine, cysteine, glycine,                              phenylalanin - #e, proline, tryptophan, tyrosine,              #and methioninenorleucine                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                - Lys Trp Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Gly         #                 15                                                          - Gly Leu Leu Ser Asn Ile Val Thr Ser Leu                                     #             25                                                              - (2) INFORMATION FOR SEQ ID NO:30:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 26 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                           #8, 10, 11, 14 and 15N: 4, 7,                                                 #where Xaa at positionsORMATION:                                              #8, 10, 11, 14 and 15 are selected from argin - #ine,                                        lysine, g - #lutamic acid, aspartic acid, glutamine,           #serine, histidine and threonine                                              #9, 12 and 13 LOCATION: 5, 6,                                                 #where Xaa at positionsORMATION:                                              #9, 12 and 13 are selected from isoleucine,                                                  valine, l - #eucine, alanine, cysteine, glycine,                              phenylalanin - #e, proline, tryptophan, tyrosine,              #and methioninenorleucine                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                - Lys Trp Lys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Gly         #                 15                                                          - Pro Ile Leu Ala Asn Leu Val Ser Ile Val                                     #             25                                                              - (2) INFORMATION FOR SEQ ID NO:31:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 20 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                           #10 and 11(B) LOCATION: 7, 8,                                                 #where Xaa at positions 7, 8, 10 and 11                                                      are selec - #ted from isoleucine, valine, leucine,             alanine,                                                                      #glycine, phenylalanine, proline, tryptophan,                                 #norleucine and methionine                                                    #and 13   (B) LOCATION: 9, 12,                                                #where Xaa at positions 9, 12, and 13                                                        are selec - #ted from arginine, lysine, glutamic acid,         #acid, glutamine, asparagine, serine,                                         #and threonine histidine                                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                - Lys Lys Trp Trp Arg Arg Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Gly Pro Ala         #                 15                                                          - Leu Ser Asn Val                                                                          20                                                               - (2) INFORMATION FOR SEQ ID NO:32:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 7 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (B) LOCATION: 7...7                                                 #where Xaa at position 7 is from                                                             about 14- - #24 amino acid residues selected from              #valine, leucine, alanine, cysteine,                                          #phenylalanine, proline, tryptophan, tyrosine,                                #methionine, arginine, lysine, glutamic                                                      acid, asp - #artic acid, glutamine, asparagine, serine,        #and threonine histidine                                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                - Lys Lys Trp Trp Arg Arg Xaa                                                   1               5                                                           - (2) INFORMATION FOR SEQ ID NO:33:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 6 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (B) LOCATION: 6...6                                                 #where Xaa at position 6 is from                                                             about 14- - #24 amino acid residues selected from              #valine, leucine, alanine, cysteine,                                          #phenylalanine, proline, tryptophan, tyrosine,                                #methionine, arginine, lysine, glutamic                                                      acid, asp - #artic acid, glutamine, asparagine, serine,        #and threonine histidine                                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                - Lys Lys Trp Trp Lys Xaa                                                       1               5                                                           - (2) INFORMATION FOR SEQ ID NO:34:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 8 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                - Lys Trp Lys Ser Phe Ile Lys Lys                                               1               5                                                           - (2) INFORMATION FOR SEQ ID NO:35:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 24 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (B) LOCATION: 6                                                     #where Xaa at position 6 is selected                                                         from argi - #nine, lysine, glutamic acid, aspartic acid,       #asparagine, serine, histidine and threonine                                  #5        (B) LOCATION: 4 and                                                 #where Xaa at positions 4 and 5 are                                           #from isoleucine, valine, leucine, alanine,                                   #glycine, phenylalanine, proline, tryptophan,                                 #norleucine and methionine                                                    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                - Lys Lys Trp Trp Arg Arg Xaa Xaa Xaa Gly Le - #u Lys Thr Ala Gly Pro         #                15                                                           - Ala Ile Gln Ser Val Leu Asn Lys                                                          20                                                               - (2) INFORMATION FOR SEQ ID NO:36:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 26 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                - Lys Trp Lys Leu Phe Lys Lys Ile Gly Ile Gl - #y Ala Val Leu Lys Val         #                15                                                           - Leu Thr Thr Gly Leu Pro Ala Leu Ile Ser                                     #             25                                                              - (2) INFORMATION FOR SEQ ID NO:37:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 28 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                - Lys Trp Lys Leu Phe Lys Lys Ile Gly Ile Gl - #y Ala Val Leu Lys Val         #                15                                                           - Leu Thr Thr Gly Leu Pro Ala Leu Lys Lys Th - #r Lys                         #             25                                                              __________________________________________________________________________

We claim:
 1. An isolated polynucleotide which encodes a peptide selectedfrom the group consisting of:

                          (SEQ ID NO:1)                                           NH.sub.2 -KWKSFIKKLTTAVKKVLTTGLPALIS-COOH,                                                          (SEQ ID NO:2)                                           NH.sub.2 -KWKSFIKKLTSAAKKVVTTAKPLISS-COOH,                                                          (SEQ ID NO:3)                                           NH.sub.2 -KWKSFIKNLTKGGSKILTTGLPALIS-COOH,                                                          (SEQ ID NO:4)                                           NH.sub.2 -KWKKFIKNLTKGGSKILTTGLPALIS-COOH,                                                          (SEQ ID NO:5)                                           NH.sub.2 -KWKSFIKNLEKVLKPGGLLSNIVTSL-COOH,                                                          (SEQ ID NO:6)                                           NH.sub.2 -KWKSFIKNLEKVLKKGPILANLVSIV-COOH,                                                          (SEQ ID NO:7)                                           NH.sub.2 -KWKEFIKKLTTAVKKVLTTGLPALIS-COOH,                                                          (SEQ ID NO:8)                                           NH.sub.2 -KWKKFIKELQKVLAPGGLLSNIVTSL-COOH,                                                          (SEQ ID NO:9)                                           NH.sub.2 -KWKSFIKKLTSVLKKVVTTALPALIS-COOH,                                                          (SEQ ID NO:10)                                          NH.sub.2 -KWKSFIKNLTKVLKKVVTTALPALIS-COOH,                                                          (SEQ ID NO:11)                                          NH.sub.2 -KWKLFKKKGTGAVLTVLTTGLPALIS-COOH,                                                          (SEQ ID NO:12)                                          NH.sub.2 -KWKSFIKKLTSVLKKVVTTAKPLISS-COOH,                                                          (SEQ ID NO:13)                                          NH.sub.2 -KKKSFIKLLTSAKVSVLTTAKPLISS-COOH,                                                          (SEQ ID NO:14)                                          NH.sub.2 -KWKKFIKELQKVLKPGGLLSNIVTSL-COOH,                                                          (SEQ ID NO:15)                                          NH.sub.2 -KKWWRRVLSGLKTGPALSNV-COOH,                                                                (SEQ ID NO:16)                                          NH.sub.2 -KKWWRRVLKGLSSGPALSNV-COOH,                                                                (SEQ ID NO:17)                                          NH.sub.2 -KKWWRRALQALKNGPALSNV-COOH,                                                                (SEQ ID NO:18)                                          NH.sub.2 -KKWWRRVLSGLKTAGPAIQSVLNK-COOH,                                                            (SEQ ID NO:19)                                          NH.sub.2 -KKWWRRALQGLKTAGPAIQSVLNK-COOH,                                                            (SEQ ID NO:20)                                          NH.sub.2 -KKWWKAQKAVNSGPNALQTLAQ-COOH,                                                              (SEQ ID NO:21)                                          NH.sub.2 -KKWWKAKKFANSGPNALQTLAQ-COOH,                                        and                                                                                                 (SEQ ID NO:22)                                          NH.sub.2 -KKWWKFIKKAVNSGTTGLQTLAS-COOH.                                   


2. An isolated polynucleotide which encodes a peptide selected from thegroup consisting of:

    NH.sub.2 -KKSFFKKLTSVASSVLS-COOH,                                                                     (SEQ ID NO:23)                                        NH.sub.2 -WKVFKSFIKKASSFAQSVLD-COOH,                                                                  (SEQ ID NO:24),                                       and                                                                           NH.sub.2 -KKWRKSFFKQVGSFDNSV-COOH.                                                                    (SEQ ID NO:25).                                   